Multipotent adult stem cells and methods for isolation

ABSTRACT

The invention provides isolated stem cells of non-embryonic origin that can be maintained in culture in the undifferentiated state or differentiated to form cells of multiple tissue types. Also provided are methods of isolation and culture, as well as therapeutic uses for the isolated cells.

Portions of the present invention were made with support of the UnitedStates Government via a grant from the National Institutes ofHealth/National Institute of Allergy and Infectious Diseases to MorayamaReyes under grant number 1F31AI-Gn10291. The U.S. Government maytherefore have certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to methods for isolation of stem cells,cells isolated by the methods, and therapeutic uses for those cells.More specifically, the invention relates to isolated marrow-derivedprogenitor cells which have the potential to differentiate to form cellsof a variety of cell lineages, as well as methods for isolating thecells and for inducing specific differentiation of the cells isolated bythe method, and specific markers that are present in these cells such asproteins and transcription factors.

BACKGROUND OF THE INVENTION

Organ and tissue generation from stem cells and their subsequenttransplantation provide promising treatments for a number ofpathologies, making stem cells a central focus of research in manyfields. Using stem cells for generation of organs and tissues fortransplantation provides a promising alternative therapy for diabetes,Parkinson's disease, liver disease, heart disease, and autoimmunedisorders, to name a few. However, there are at least two major problemsassociated with organ and tissue transplantation. First, there is ashortage of donor organs and tissues. As few as 5 percent of the organsneeded for transplant in the United States along ever become availableto a recipient. (Evans, et al., J. Am. Med. Assoc. (1992) 267: 239-246.)According to the American Heart Association, only 2,300 of the 40,000Americans who needed a new heart in 1997 received one, and the AmericanLiver Foundation reports that there are fewer than 3,000 donors for thenearly 30,000 patients who die each year from liver failure. The secondmajor problem is the potential incompatibility of the transplantedtissue with the immune system of the recipient. Because the donatedorgan or tissue is recognized by the host immune system as foreign,anti-rejection medications must be provided to the patient at asignificant cost—both financially and physically.

Xenotransplantation, or transplantation of tissue or organs from anotherspecies, could provide an alternative means to overcome the shortage ofhuman organs and tissues. Xenotransplantation would offer the advantageof advanced planning of the transplant, allowing the organ to beharvested while still healthy and allowing the patient to undergo anybeneficial pretreatment prior to transplant surgery. Unfortunately,xenotransplantation does not overcome the problem of tissueincompatibility, but instead exacerbates it. Furthermore, according tothe Centers for Disease Control, there is evidence that damaging virusescross species barriers. Pigs have become likely candidates as organ andtissue donors, yet cross-species transmission of more than one virusfrom pigs to humans has been documented. For example, over a millionpigs were recently slaughtered in Malaysia in an effort to contain anoutbreak of Hendra virus, a disease that was transmitted to more than 70humans with deadly results. (Butler, D., Nature (1999) 398: 549.)

Stem Cells: Definition and Use

The most promising source of organs and tissues for transplantationtherefore lies in the development of stem cell technology.Theoretically, stem cells can undergo self-renewing cell division togive rise to phenotypically and genotypically identical daughters for anindefinite time and ultimately can differentiate into at least one finalcell type. By generating tissues or organs from a patient's own stemcells, or by genetically altering heterologous cells so that therecipient immune system does not recognize them as foreign, transplanttissues can be generated to provide the advantages associated withxenotransplantation without the associated risk of infection or tissuerejection.

Stem cells also provide promise for improving the results of genetherapy. A patient's own stem cells could be genetically altered invitro, then reintroduced in vivo to produce a desired gene product.These genetically altered stem cells would have the potential to beinduced to differentiate to form a multitude of cell types forimplantation at specific sites in the body, or for systemic application.Alternately, heterologous stem cells could be genetically altered toexpress the recipient's major histocompatibility complex (MHC) antigen,or no MHC, to allow transplant of those cells from donor to recipientwithout the associated risk of rejection.

Stem cells are defined as cells that have extensive, some would sayindefinite, proliferation potential that differentiate into several celllineages, and that can repopulate tissues upon transplantation. Thequintessential stem cell is the embryonal stem (ES) cell, as it hasunlimited self-renewal and multipotent differentiation potential. Thesecells are derived from the inner cell mass of the blastocyst, or can bederived from the primordial germ cells from a post-implantation embryo(embryonal germ cells or EG cells). ES and EG cells have been derivedfrom mouse, and more recently also from non-human primates and humans.When introduced into mouse blastocysts or blastocysts of other animals,ES cells can contribute to all tissues of the mouse (animal). Whentransplanted in post-natal animals, ES and EG cells generate teratomas,which again demonstrates their multipotency. ES (and EG) cells can beidentified by positive staining with the antibodies SSEA1 and SSEA4.

At the molecular level, ES and EG cells express a number oftranscription factors highly specific for these undifferentiated cells.These include oct-4 and Rex-1. Also found are the LIF-R and thetranscription factors sox-2 and Rox-1, even though the latter two arealso expressed in non-ES cells. oct-4 is a transcription factorexpressed in the pregastrulation embryo, early cleavage stage embryo,cells of the inner cell mass of the blastocyst, and in embryoniccarcinoma (EC) cells. oct-4 is down-regulated when cells are induced todifferentiate in vitro and in the adult animal oct-4 is only found ingerm cells. Several studies have shown that oct-4 is required formaintaining the undifferentiated phenotype of ES cells, and plays amajor role in determining early steps in embryogenesis anddifferentiation. oct-4, in combination with Rox-1, causestranscriptional activation of the Zn-finger protein Rex-1, and is alsorequired for maintaining ES in an undifferentiated state. Likewise,sox-2, is needed together with oct-4 to retain the undifferentiatedstate of ES/EC and to maintain murine (but not human) ES cells. Human ormurine primordial germ cells require presence of LIF. Another hallmarkof ES cells is presence of telomerase, which provides these cells withan unlimited self-renewal potential in vitro.

Stem cells have been identified in most organ tissues. The bestcharacterized is the hematopoietic stem cell. This is a mesoderm-derivedcell that has been purified based on cell surface markers and functionalcharacteristics. The hematopoietic stem cell, isolated from bone marrow,blood, cord blood, fetal liver and yolk sac, is the progenitor cell thatreinitiates hematopoiesis for the life of a recipient and generatesmultiple hematopoietic lineages (see Fei, R., et al., U.S. Pat. No.5,635,387; McGlave, et al., U.S. Pat. No. 5,460,964; Simmons, P., etal., U.S. Pat. No. 5,677,136; Tsukamoto, et al., U.S. Pat. No.5,750,397; Schwartz, et al., U.S. Pat. No. 759,793; DiGuisto, et al.,U.S. Pat. No. 5,681,599; Tsukamoto, et al., U.S. Pat. No. 5,716,827;Hill, B., et al., Exp. Hematol. (1996) 24 (8): 936-943). Whentransplanted into lethally irradiated animals or humans, hematopoieticstem cells can repopulate the erythroid, neutrophil-macrophage,megakaryocyte and lymphoid hemopoietic cell pool. In vitro, hemopoieticstem cells can be induced to undergo at least some self-renewing celldivisions and can be induced to differentiate to the same lineages as isseen in vivo. Therefore, this cell fulfills the criteria of a stem cell.Stem cells which differentiate only to form cells of hematopoieticlineage, however, are unable to provide a source of cells for repair ofother damaged tissues, for example, heart or lung tissue damaged byhigh-dose chemotherapeutic agents.

A second stem cell that has been studied extensively is the neural stemcell (Gage F H: Science 287:1433-1438, 2000; Svendsen C N et al, BrainPath 9:499-513, 1999; Okabe S et al, Mech Dev 59:89-102, 1996). Neuralstem cells were initially identified in the subventricular zone and theolfactory bulb of fetal brain. Until recently, it was believed that theadult brain no longer contained cells with stem cell potential. However,several studies in rodents, and more recently also non-human primatesand humans, have shown that stem cells continue to be present in adultbrain. These stem cells can proliferate in vivo and continuouslyregenerate at least some neuronal cells in vivo. When cultured ex vivo,neural stem cells can be induced to proliferate, as well as todifferentiate into different types of neurons and glial cells. Whentransplanted into the brain, neural stem cells can engraft and generateneural cells and glial cells. Therefore, this cell too fulfills thedefinition of a stem cell.

Mesenchymal stem cells (MSC), originally derived from the embryonalmesoderm and isolated from adult bone marrow, can differentiate to formmuscle, bone, cartilage, fat, marrow stroma, and tendon. Duringembryogenesis, the mesoderm develops into limb-bud mesoderm, tissue thatgenerates bone, cartilage, fat, skeletal muscle and possiblyendothelium. Mesoderm also differentiates to visceral mesoderm, whichcan give rise to cardiac muscle, smooth muscle, or blood islandsconsisting of endothelium and hematopoietic progenitor cells. Primitivemesodermal or mesenchymal stem cells, therefore, could provide a sourcefor a number of cell and tissue types. A third tissue specific cell thathas been named a stem cell is the mesenchymal stem cell, initiallydescribed by Fridenshtein (Fridenshtein, Arkh. Patol., 44:3-11, 1982). Anumber of mesenchymal stem cells have been isolated (see, for example,Caplan, A., et al., U.S. Pat. No. 5,486,359; Young, H., et al., U.S.Pat. No. 5,827,735; Caplan, A., et al., U.S. Pat. No. 5,811,094; Bruder,S., et al., U.S. Pat. No. 5,736,396; Caplan, A., et al., U.S. Pat. No.5,837,539; Masinovsky, B., U.S. Pat. No. 5,837,670; Pittenger, M., U.S.Pat. No. 5,827,740; Jaiswal, N., et al., J. Cell Biochem. (1997) 64(2):295-312; Cassiede P., et al., J. Bone Miner. Res. (1996) 11(9):1264-1273; Johnstone, B., et al., (1998) 238(1): 265-272; Yoo, et al.,J. Bone Joint Surg. Am. (1998) 80(12): 1745-1757; Gronthos, S., Blood(1994) 84(12): 4164-4173; Makino, S., et al., J. Clin. Invest. (1999)103(5): 697-705). Of the many mesenchymal stem cells that have beendescribed, all have demonstrated limited differentiation to form onlythose differentiated cells generally considered to be of mesenchymalorigin. To date, the most multipotent mesenchymal stem cell reported isthe cell isolated by Pittenger, et al., which expresses the SH2⁺ SH4⁺CD29⁺ CD44⁺ CD71⁺ CD90⁺ CD106⁺ CD120a⁺ CD124⁺ CD14⁻ CD34⁻ CD45⁻phenotype. This cell is capable of differentiating to form a number ofcell types of mesenchymal origin, but is apparently limited indifferentiation potential to cells of the mesenchymal lineage, as theteam who isolated it noted that hematopoietic cells were neveridentified in the expanded cultures. (Pittenger, et al., Science (1999)284: 143-147.)

Other stem cells have been identified, including gastrointestinal stemcells, epidermal stem cells, and hepatic stem cells, also termed ovalcells (Potten C, Philos Trans R Soc Lond B Biol Sci 353:821-30, 1998;Watt F, Philos. Trans R Soc Lond B Biol Sci 353:831, 1997; Alison M etal, Hepatol 29:678-83, 1998). Most of these are less well characterized.

Compared with ES cells, tissue specific stem cells have lessself-renewal ability and, although they differentiate into multiplelineages, they are not multipotent. No studies have addressed whethertissue specific cells express markers described above of ES cells. Inaddition, the degree of telomerase activity in tissue specific stemcells has not been fully explored, in part because large numbers ofhighly enriched populations of these cells are difficult to obtain.

Until recently, it was thought that organ specific stem cells could onlydifferentiate into cells of the same tissue. A number of recentpublications have suggested that adult organ specific stem cells may becapable of differentiating into cells of different tissues. A number ofstudies have shown that cells transplanted at the time of a bone marrowtransplant can differentiate into skeletal muscle (Ferrari Science279:528-30, 1998; Gussoni Nature 401:390-4, 1999). This could beconsidered within the realm of possible differentiation potential ofmesenchymal cells that are present in marrow. Jackson published thatmuscle satellite cells can differentiate into hemopoietic cells, again aswitch in phenotype within the splanchnic mesoderm (Jackson PNAS USA96:14482-6, 1999). Other studies have shown that stem cells from oneembryonal layer (for instance splanchnic mesoderm) can differentiateinto tissues thought to be derived during embryogenesis from a differentembryonal layer. For instance, endothelial cells or their precursorsdetected in humans or animals that underwent marrow transplantation areat least in part derived from the marrow donor (Takahashi, Nat Med5:434-8, 1999; Lin, Clin Invest 105:71-7, 2000). Thus, visceral mesodermand not splanchnic mesoderm, such as MSC, derived progeny aretransferred with the infused marrow. Even more surprising are thereports demonstrating both in rodents and humans that hepatic epithelialcells and biliary duct epithelial cells are derived from the donormarrow (Petersen, Science 284:1168-1170, 1999; Theise, Hepatology31:235-40, 2000; Theise, Hepatology 32:11-6, 2000). Likewise, threegroups have shown that neural stem cells can differentiate intohemopoietic cells. Finally, Clarke et al. reported that neural stemcells injected into blastocysts can contribute to all tissues of thechimeric mouse (Clarke, Science 288:1660-3, 2000).

It is necessary to point out that most of these studies have notconclusively demonstrated that a single cell can differentiate intotissues of different organs. Indeed most investigators did not identifythe phenotype of the initiating cell. An exception is the study byWeissman and Grompe, who showed that cells that repopulated the liverwere present in Lin⁻Thy₁LowSca₁+ marrow cells, which are highly enrichedin hematopoietic stem cells. Likewise, the Mulligan group showed thatmarrow Sp cells, highly enriched for HSC, can differentiate into muscleand endothelium, and Jackson et al. showed that muscle Sp cells areresponsible for hemopoietic reconstitution (Gussoni et al., Nature401:390-4, 1999).

Transplantation of tissues and organs generated from heterologousembryonic stem cells requires either that the cells be furthergenetically modified to inhibit expression of certain cell surfacemarkers, or that the use of chemotherapeutic immune suppressors continuein order to protect against transplant rejection. Thus, althoughembryonic stem cell research provides a promising alternative solutionto the problem of a limited supply of organs for transplantation, theproblems and risks associated with the need for immunosuppression tosustain transplantation of heterologous cells or tissue would remain. Anestimated 20 immunologically different lines of embryonic stem cellswould need to be established in order to provide immunocompatible cellsfor therapies directed to the majority of the population (Wadman, M.,Nature (1999) 398: 551).

Using cells from the developed individual, rather than an embryo, as asource of autologous or allogeneic stem cells would overcome the problemof tissue incompatibility associated with the use of transplantedembryonic stem cells, as well as solve the ethical dilemma associatedwith embryonic stem cell research. The greatest disadvantage associatedwith the use of autologous stem cells for tissue transplant thus farlies in their limited differentiation potential. A number of stem cellshave been isolated from fully-developed organisms, particularly humans,but these cells, although reported to be multipotent, have demonstratedlimited potential to differentiate to multiple cell types.

Thus, even though stem cells with multiple differentiation potentialhave been isolated previously by others and by the present inventors, aprogenitor cell with the potential to differentiate into a wide varietyof cell types of different lineages, including fibroblasts, osteoblasts,chondrocytes, adipocytes, skeletal muscle, endothelium, stroma, smoothmuscle, cardiac muscle and hemopoietic cells, has not been described. Ifcell and tissue transplant and gene therapy are to provide thetherapeutic advances expected, a stem cell or progenitor cell with thegreatest or most extensive differentiation potential is needed. What isneeded is the adult equivalent of an embryonic stem cell.

SUMMARY OF THE INVENTION

The present invention provides an isolated multipotent mammalian stemcell that is surface antigen negative for CD44, CD45, and HLA Class Iand II. The cell may also be surface antigen negative for CD34, Muc18,Stro-1, HLA-class-I and may be positive for oct3/4 mRNA, and may bepositive for hTRT mRNA. In particular, the cell may be surface antigennegative for CD31, CD34, CD36, CD38, CD45, CD50, CD62E and CD62P,HLA-DR, Muc18, STRO-1, cKit, Tie/Tek, CD44, HLA-class 1 and2-microglobulin and is positive for CD10, CD13, CD49b, CD49e, CDw90,Flk1, EGF-R, TGF-R1 and TGF-R2, BMP-R1A, PDGF-R1a and PDGF-R1b. Thepresent invention provides an isolated multipotent non-embryonic,non-germ cell line cell that expresses transcription factors oct3/4,REX-1 and ROX-1. It also provides an isolated multipotent cell derivedfrom a post-natal mammal that responds to growth factor LIF and hasreceptors for LIF.

The cells of the present invention described above may have the capacityto be induced to differentiate to form at least one differentiated celltype of mesodermal, ectodermal and endodermal origin. For example, thecells may have the capacity to be induced to differentiate to form cellsof at least osteoblast, chondrocyte, adipocyte, fibroblast, marrowstroma, skeletal muscle, smooth muscle, cardiac muscle, endothelial,epithelial, hematopoietic, glial, neuronal or oligodendrocyte cell type.The cell may be a human cell or a mouse cell. The cell may be from afetus, newborn, child, or adult. The cell may be derived from an organ,such as from marrow, liver or brain.

The present invention further provides differentiated cells obtainedfrom the multipotent adult stem cell described above, wherein theprogeny cell may be a bone, cartilage, adipocyte, fibroblast, marrowstroma, skeletal muscle, smooth muscle, cardiac muscle, endothelial,epithelial, endocrine, exocrine, hematopoietic, glial, neuronal oroligodendrocyte cell. The differentiated progeny cell may be a skinepithelial cell, liver epithelial cell, pancreas epithelial cell,pancreas endocrine cell or islet cell, pancreas exocrine cell, gutepithelium cell, kidney epithelium cell, or an epidermal associatedstructure (such as a hair follicle). The differentiated progeny cell mayform soft tissues surrounding teeth or may form teeth.

The present invention provides an isolated transgenic multipotentmammalian stem cell as described above, wherein genome of the cell hasbeen altered by insertion of preselected isolated DNA, by substitutionof a segment of the cellular genome with preselected isolated DNA, or bydeletion of or inactivation of at least a portion of the cellulargenome. This alteration may be by viral transduction, such as byinsertion of DNA by viral vector integration, or by using a DNA virus,RNA virus or retroviral vector. Alternatively, a portion of the cellulargenome of the isolated transgenic cell may be inactivated using anantisense nucleic acid molecule whose sequence is complementary to thesequence of the portion of the cellular genome to be inactivated.Further, a portion of the cellular genome may be inactivated using aribozyme sequence directed to the sequence of the portion of thecellular genome to be inactivated. The altered genome may contain thegenetic sequence of a selectable or screenable marker gene that isexpressed so that the progenitor cell with altered genome, or itsprogeny, can be differentiated from progenitor cells having an unalteredgenome. For example, the marker may be a green, red, yellow fluorescentprotein, Beta-gal, Neo, DHFR^(m), or hygromycin. The cell may express agene that can be regulated by an inducible promoter or other controlmechanism to regulate the expression of a protein, enzyme or other cellproduct.

The present invention provides a cell that may express high levels oftelomerase and may maintain long telomeres after extended in vitroculture, as compared to the telomeres from lymphocytes from the samedonors. The telomeres may be about 11-16 KB in length after extended invitro culture.

The present invention provides a cell differentiation solutioncomprising factors that modulate the level of oct3/4 expression forpromoting continued growth or differentiation of undifferentiatedmultipotent stem cells.

The present invention provides a method for isolating multipotent adultstem cells (MASC). The method involves depleting bone marrow mononuclearcells of CD45⁺ glycophorin A⁺ cells, recovering CD45− glycophorin A−cells, plating the recovered CD45− glycophorin A− cells onto a matrixcoating, and culturing the plated cells in media supplemented withgrowth factors. The step of depleting may involved negative selectionusing monoclonal or polyclonal antibodies. The growth factors may bechosen from PDGF-BB, EGF, IGF, and LIF. The last step may furtherinvolve culturing in media supplemented with dexamethasone, linoleicacid, and/or ascorbic acid.

The present invention provides a culture method for isolatingmultipotent adult stem cells involving adding the cells to serum-free orlow-serum medium containing insulin, selenium, bovine serum albumin,linoleic acid, dexamethasone, and platelet-derived growth factor. Theserum-free or low-serum medium may be low-glucose DMEM in admixture withMCDB. The insulin may be present at a concentration of from about 10 toabout 50 μg/ml. The serum-free or low-serum medium may contain aneffective amount of transferrin at a concentration of greater than 0 butless than about 10 μg/ml, the selenium may be present at a concentrationof about 0.1 to about 5 μg/ml, the bovine serum albumin may be presentat a concentration of about 0.1 to about 5 μg/ml, the linoleic acid maybe present at a concentration of about 2 to about 10 μg/m, and thedexamethasone may be present at a concentration of about 0.005 to 0.15μM. The serum-free medium or low-serum medium may contain about 0.05-0.2mM L-ascorbic acid. The serum-free medium or low-serum medium maycontain about 5 to about 15 ng/ml platelet-derived growth factor, 5 toabout 15 ng/ml epidermal growth factor, 5 to about 15 ng/ml insulin-likegrowth factor, 10-10,000 IU leukemia inhibitory factor. The presentinvention further provides a cultured clonal population of mammalianmultipotent adult stem cells isolated according to the above-describedmethod.

The present invention provides a method to permanently and/orconditionally immortalize MASC derived cells and differentiated progenyby transferring telomerase into MASC or differentiated progeny.

The present invention provides a method to reconstitute thehematopoietic and immune system of a mammal by administering to themammal fully allogenic multipotent stem cells (MASC), derivedhematopoietic stem cells, or progenitor cells to induce tolerance in themammal for subsequent multipotent stem cell derived tissue transplantsor other organ transplants.

The present invention provides a method of expanding undifferentiatedmultipotent stem cells into differentiated hair follicles byadministering appropriate growth factors, and growing the cells.

The present invention provide numerous uses for the above-describedcells. For example, the invention provides a method of using theisolated cells by performing an in utero transplantation of a populationof the cells to form chimerism of cells or tissues, thereby producinghuman cells in prenatal or post-natal humans or animals followingtransplantation, wherein the cells produce therapeutic enzymes,proteins, or other products in the human or animal so that geneticdefects are corrected. The present invention also provides a method ofusing the cells for gene therapy in a subject in need of therapeutictreatment, involving genetically altering the cells by introducing intothe cell an isolated pre-selected DNA encoding a desired gene product,expanding the cells in culture, and introducing the cells into the bodyof the subject to produce the desired gene product.

The present invention provides a method of repairing damaged tissue in ahuman subject in need of such repair by expanding the isolatedmultipotent adult stem cells in culture, and contacting an effectiveamount of the expanded cells with the damaged tissue of said subject.The cells may be introduced into the body of the subject by localizedinjection, or by systemic injection. The cells may be introduced intothe body of the subject in conjunction with a suitable matrix implant.The matrix implant may provide additional genetic material, cytokines,growth factors, or other factors to promote growth and differentiationof the cells. The cells may be encapsulated prior to introduction intothe body of the subject, such as within a polymer capsule.

The present invention provides a method for inducing an immune responseto an infectious agent in a human subject involving genetically alteringan expanded clonal population of multipotent adult stem cells in cultureexpress one or more pre-selected antigenic molecules that elicit aprotective immune response against an infectious agent, and introducinginto the subject an amount of the genetically altered cells effective toinduce the immune response. The present method may further involve,prior to the second step, the step of differentiating the multipotentadult stem cells to form dendritic cells.

The present invention provides a method of using MASCs to identifygenetic polymorphisms associated with physiologic abnormalities,involving isolating the MASCs from a statistically significantpopulation of individuals from whom phenotypic data can be obtained,culture expanding the MASCs from the statistically significantpopulation of individuals to establish MASC cultures, identifying atleast one genetic polymorphism in the cultured MASCs, inducing thecultured MASCs to differentiate, and characterizing aberrant metabolicprocesses associated with said at least one genetic polymorphism bycomparing the differentiation pattern exhibited by an MASC having anormal genotype with the differentiation pattern exhibited by an MASChaving an identified genetic polymorphism.

The present invention further provides a method for treating cancer in amammalian subject involving genetically altering multipotent adult stemcells to express a tumoricidal protein, an anti-angiogenic protein, or aprotein that is expressed on the surface of a tumor cell in conjunctionwith a protein associated with stimulation of an immune response toantigen, and introducing an effective anti-cancer amount of thegenetically altered multipotent adult stem cells into the mammaliansubject.

The present invention provides a method of using MASCs to characterizecellular responses to biologic or pharmacologic agents involvingisolating MASCs from a statistically significant population ofindividuals, culture expanding the MASCs from the statisticallysignificant population of individuals to establish a plurality of MASCcultures, contacting the MASC cultures with one or more biologic orpharmacologic agents, identifying one or more cellular responses to theone or more biologic or pharmacologic agents, and comparing the one ormore cellular responses of the MASC cultures from individuals in thestatistically significant population.

The present invention also provides a method of using specificallydifferentiated cells for therapy comprising administering thespecifically differentiated cells to a patient in need thereof. Itfurther provides for the use of genetically engineered multipotent stemcells to selectively express an endogenous gene or a transgene, and forthe use of MASCs grown in vivo for transplantation/administration intoan animal to treat a disease. For example, neuroretinal cells derivedfrom multipotent stem or MASCs can be used to treat blindness caused byamong other things but not limited to neuroretinal disease caused byamong other things macular degeneration, diabetic retinopathy, glaucoma,retinitis pigmentosa. The cells can be used to engraft a cell into amammal comprising administering autologous, allogenic or xenogeniccells, to restore or correct tissue specific metabolic, enzymatic,coagulation, structural or other function to the mammal. The cells canbe used to engraft a cell into a mammal, causing the differentiation invivo of cell types, and for administering the differentiated stem cellsinto the mammal. The cells, or their in vitro or in vivo differentiatedprogeny, can be used to correct a genetic disease, degenerative disease,cardiovascular disease, metabolic storage disease, neural, or cancerdisease process. They can be used to produce gingiva-like material fortreatment of periodontal disease. They can be used to develop skinepithelial tissue derived from multipotent stem cells that can beutilized for skin grafting and plastic surgery. They could be used toenhance muscle such as in the penis or heart. The can be used to produceblood ex vivo for therapeutic use, or to produce human hematopoieticcells and/or blood in prenatal or post natal animals for human use. Theycan be used as a therapeutic to aid for example in the recovery of apatient from chemotherapy or radiation therapy in treatment of cancer,in the treatment of autoimmune disease, to induce tolerance in therecipient. They can be used to treat AIDS or other infectious diseases.

The cardiomyocytes or MASC can be used to treat cardiac diseasesincluding among others but not limited to myocarditis, cardiomyopathy,heart failure, damage caused by heart attacks, hypertension,atherosclerosis, heart valve dysfunction. A genetically engineeredmultipotent mammalian derived stem cell, or its differentiated progeny,can be used to treat a disease with CNS deficits or damage. Further themultipotent mammalian derived stem cell, or its neuronally relateddifferentiated cell, can be used to treat a disease with neural deficitsor degeneration including among but not limited to stroke, Alzhemier's,Parkinson's disease, Huntington's disease, AIDS associated dementia,spinal cord injury, metabolic diseases effecting the brain or othernerves.

A multipotent mammalian derived stem cell or their differentiatedprogeny such as stromal cells can be used to support the growth anddifferentiation of other cell types in vivo or in vitro, including butnot limited to hematopoietic cells, pancreatic islet or beta cells,hepatocytes, etc. The stem cell, or cartilage differentiated progeny,can be used to treat a disease of the joints or cartilage including butnot limited to cartilage tears, cartilage thinning, osteoarthritis.Moreover, the stem cells or their osteoblast differentiated progeny canbe used to ameliorate a process having deleterious effects on boneincluding among but not limited to bone fractures, non-healingfractures, osteoarthritis, “holes” in bones cause by tumors spreading tobone such as prostate, breast, multiple myloma etc.

The present invention also provides a kit for providing immunization toinduce a protective immune response in a human subject. The kit maycontain, separately packaged, media and antibodies for isolation ofmultipotent adult stem cells from a bone marrow aspirate; media andcellular factors for culture of the isolated multipotent adult stemcells; and genetic elements for genetically altering the multipotentadult stem cells to produce antigenic molecules. The kit may furthercontain media and cellular factors effective to differentiate themultipotent adult stem cells to form tissue-specific cell types. Thegenetic elements may be viral vectors, and the viral vectors may containthe nucleotide sequence encoding one or more antigens of bacterial orviral origin. The genetic elements may be plasmids containing anucleotide sequence encoding a bacterial, viral, or parasite antigen.The plasmids may be packaged with components for calcium phosphatetransfection. The genetic elements may be vectors comprising nucleotidesequences encoding antigens common to cancer cells, or the geneticelements may be vectors containing nucleotide sequences encodingantigens of parasitic organisms.

The present invention further provides a method of gene profiling of amultipotent derived stem cell as described above, and the use of thisgene profiling in a data bank. It also provides for the use of geneprofiled multipotent stem cells as described above in data bases to aidin drug discovery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a and FIG. 1 b are photographs of undifferentiated MASCs of thepresent invention. Cells lacking CD45 expression, as well asglycophorin-A expression were selected by immunomagnetic bead depletionand FACS. Cells recovered after sorting are small blasts (FIG. 1 a).5000 cells were plated in fibronectin coated wells of 96 well plates indefined medium consisting of DMEM, 10 ng/ml IGF, 10 ng/ml EGF and 10ng/ml PDGF-BB as well as transferrin, selenium, bovine serum albumin,dexamethasone, linoleic acid, insulin and ascorbic acid. After 7-21days, small colonies of adherent cells develop. (FIG. 1 b).

FIG. 2 is a graph illustrating expansion rates for MASCs in culture.CD45−/GlyA− cells were plated in fibronectin-coated wells of 96 wellplates in defined medium consisting of DMEM, 10 ng/ml IGF, 10 ng/ml EGFand 10 ng/ml PDGF-BB as well as transferrin, selenium, bovine serumalbumin, dexamethasone, linoleic acid, insulin and ascorbic acid with orwithout 2% FCS. When semi-confluent, cells were recovered bytrypsinization and sub-cultured twice weekly at a 1:4 dilution under thesame culture conditions.

FIG. 3 Telomere length of MASCS from a donor, age 35, was cultured atreseeding densities of 2×10³ cells/cm² for 23 and 35 cell doublings.Telomere length was determined using standard techniques. Telomerelength was 9 kB. This was 3 kB longer than telomere length of bloodlymphocytes obtained from the same donor. Telomere length evaluatedafter 10 and 25 cell doublings resp. and again after 35 cells doublings,was unchanged. As controls, we tested HL60 cells (short telomeres) and293 cells (long telomeres).

FIG. 4 illustrates the general protocol for culture, transduction,differentiation, and confirmation of differentiation used by theinventors for MASCs of the present invention. Transduction with aneGFP-containing retroviral vector was performed after culture asindicated. Half-confluent MASC were exposed for six hours on twosequential days to MFG-eGFP containing PA317 supernatant made in MASCmedium (i.e., DMEM, 2% FCS, EGF, PDGF-BB, transferrin, selenium, bovineserum albumin, dexamethasone, linoleic acid, insulin and ascorbic acid)in the presence of 10 μg/mL protamine. Twenty-four hours after the lasttransduction, cells were trypsinized and subjected to FACS selection.Thirty to seventy percent of MASCs were eGFP positive. One to onehundred eGFP positive cells/well were sorted using the ACDU device onthe FACS in FN coated wells of 96 well plates, in the same MASC medium.Of these wells, approximately 2/plate containing 10 cells/well producedMASC progeny. Clones were then culture expanded. Eight to 10sub-populations of these expanded cells were induced to differentiatealong different pathways, with differentiation being confirmed using thetechniques indicated.

FIG. 5 illustrates the differentiation protocol used by the inventors toinduce the MASCs of the present invention to differentiate to formosteoblasts, chondroblasts and adipocytes as indicated. Depicted are thecytokines needed and the appropriate tests to demonstrate induction ofterminal differentiation.

FIG. 6. illustrates results of immunohistochemistry staining for bonesialoprotein on day 15 as well as Western blot analysis for bonesialoprotein on days 7, 11 and 14 after culture after induction of MASCswith 10⁻⁷M dexamethasone, β-glycerophosphate and 10 mM ascorbic acid. Inthe middle panel, results of toluidin blue staining for cartilage aswell as Western blot analysis for collagen type II on days 7, 11 and 14shows differentiation to chondrocytes following culture of MASCs inmicromass in serum free medium with 100 ng./mL TGF-β1. In the lowerpanel, oil-red staining on day 14 and Western blot analysis for PPARgshows differentiation following treatment of MASCs with 10% horse serum.

FIG. 7. shows Western blot analysis for muscle proteins. Panel A, showsresults of culture of confluent MASCs with 3 μM 5-azacytidine for 24 h.Cultures were then maintained in MASC expansion medium (DMEM, 2% FCS,EGF, PDGF-BB, transferrin, selenium, bovine serum albumin,dexamethasone, linoleic acid, insulin and ascorbic acid).Differentiation was evaluated by Western blot. 5 days after inductionwith either 5-azacytidine, the Myf5, Myo-D and Myf6 transcriptionfactors could be detected in approximately 50% of cells. After 14-18days, Myo-D was expressed at significantly lower levels, whereas Myf5and Myf6 persisted. We detected desmin and skeletal actin as early as 4days after induction, and skeletal myosin at 14 days. Byimmunohistochemistry, 70-80% of cells expressed mature muscle proteinsafter 14 days (not shown). Treatment with either 5-azacytidine orretinoic acid resulted in expression of Gata4 and Gata6 during the firstweek of culture. In addition, low levels of troponin-T could be detectedfrom day 2 on, which may suggest that fetal muscle generated as cardiactroponin-T is found in embryonal skeletal muscle. Smooth muscle actinwas detected at 2 days after induction and persisted till 14 days. Inpanel B, we added 100 ng/mL PDGF as the sole cytokine to confluent MASCSmaintained in serum-free medium for 14 days. Presence of smooth musclemarkers was evaluated by Western blot. Smooth muscle actin was detectedfrom day 2 on and smooth muscle myosin after 6 days. Approximately 70%of cells stained positive with anti-smooth muscle actin and myosinantibodies on day 15 by immunohistochemistry. We found presence ofmyogenin from day 4 on and desmin after 6 days. We also detected Myf5and Myf6 proteins after 2-4 days, which persisted till day 15. No Myo-Dwas detected.

In panel C, confluent MASCS were exposed to retinoic acid and thencultured in serum-free medium with 100 ng/mL bFGF. Cells were thenanalyzed by Western blot. Gata4 and Gata6 were expressed as early as day2 and persisted till day 15. Cardiac troponin-T was expressed after day4 and cardiac troponin-I from day 6 on, while we could detect ANP afterday 11 (not shown). These cardiac proteins were detected in >70% ofcells by immuno-histochemistry on day 15 (not shown). We found thetranscription factor Myf6 from day 2 on. Expression of desmin started onday 6 and myogenin on day 2. We also found skeletal actin. When thecultures were maintained for >3 weeks, cells formed syncithia. We alsosaw infrequent spontaneous contractions occurring in the cultures, whichwere propagated over several mm distance.

FIG. 8 is a photomicrograph showing fusion of myoblasts and myotubes toform multinucleated myotubes. Myoblasts from an eGFP transducedpopulation of MASC subsequently induced with 5-azacytidin for 24 andmaintained in MASC expansion medium were cocultured with myoblastsgenerated from non e-GFP transduced MASCS from the same donor. To inducemyotubes, MASC derived myoblasts 9 obtained after induction ofnon-transduced MASC with 5-azacytidin for 24 h after which they weremaintained in MASC expansion medium for 14 days) were cultured with 10%horse serum in DMEM. Once multinucleated cells were formed, myotubeswere incubated with PKH26 (a red membrane dye), washed and coculturedwith eGFP transduced myotubes generated as described above in thepresence of 10% horse serum. After 2 days, cells were examined under anfluorescence microscope. The photomicrograph shows that the eGFPpositive myoblast has fused with the PKH26 labeled myotube.

FIG. 9 is a cartoon depicting methods used by the inventors to induceendothelium differentiation from MASCs of the present invention andmarkers used to detect endothelium differentiation.

FIG. 10 is a series of photographs of immunofluorescence staining forvon Willebrand factor and CD34 markers as well as a Western blotanalysis for the endothelial cell surface receptor Tie/Tek to confirmendothelial cell differentiation. MASCs express Flk1 but not CD34,PECAM, E- and P-selectin, CD36, Tie/Tek or Flt1. When MASCs werecultured serum-free MASCs medium with 20 ng/mL VEGF we saw theappearance of CD34 on the cell surface and cells expressed vWF by day 14(immuno-fluorescence). In addition, cells expressed Tie/Tek, as shown onWestern blot analysis on days 7, 11 and 14. When VEGF induced cells werecultured on matrigel or collagen type IV, vascular tube formation wasseen.

FIG. 11 is a series of photomicrographs showing that MASCs differentiateto astrocytes, oligodendrocytes and neural cells when cultured with SCF,Flt3-L, Tpo and Epo for 14 days after which they were cultured in SCFand EGF containing MASC medium by the hematopoietic supportive feederAFT024. Cells were labeled with antibodies againstglial-fibrilar-acidic-protein (GFAP) (astrocytes), galactocerebroside(GalC) (oligodendrocytes) and neurofilament-68 and 200 (neurons).

FIG. 12 is a series of photomicrographs showing that when low densityMASCs are cultured in fibronectin coated wells with 100 ng/mL bFGF,neurons develop. 20±2% cells stained positive for β-tubulin-III, 22±3%for neurofilament-68, 50±3% for neurofilament-160, 20±2% forneurofilament-200, 82±5% for neuron-specific-enolase (NSE) and 80±2% formicrotubule-assocaited-protein-2 (MAP2). The number of neurites perneuron increased from 3±1, to 5±1 and 7±2 from 2, 3 to 4 weeks afterdifferentiation. Not shown, after 2 weeks in culture, 26±4% of cellswere GFAP positive, 28±3% GalC positive, whereas fewer cells were GFAPor GalC positive after 4 weeks.

FIG. 13 shows RT-PCR results and Western blot analysis for GFAP, myelinbasic protein (MBP) and neurofilament-200x, x and days after inductionof MASCs with bFGF.

FIG. 14 shows effect of 100 ng/mL bFGF, or 10 ng/mL of either FGF-9,FGF-8, FGF-10, FGF-4, BDNF, GDNF, or CNTF on neural development fromMASCs. The nature of the differentiated cells was identified byimmunohistochemistry using antibodies agiants GFAP, GalC, neurofilament200, tyrosine hydroxylase (TH), GABA and parvalbumine, and acetylcholine(CAT). When cultured for 3 weeks with bFGF, MASC differentiated intoneurons, astrocuytes and oligodendrocytes. We did not detect GABA,parvalbumin, tyrosine hydroxylase, DOPA-decarboxylase, or tryptophanhydroxylase. When cultured for 3 weeks with long/mL FGF-9 and EGF MASCsgenerated astrocytes, oligodendrocytes and GABAergic and dopaminergic.When MASCs were cultured with 10 ng/mL FGF-8 and EGF for 3 weeks bothdopaminergic and GABAergic neurons were produced. Culture of MASCs in 10ng/mL FGF-10 and EGF for three weeks generated astrocytes andoligodendrocytes, but not neurons. When treated with 10 ng/mL FGF-4 andEGF for 3 weeks MASCs differentiated into astrocytes andoligodendrocytes but not neurons. When MASCs were treated with 10 ng/mLBDNF and EGF exclusive differentiation into tyrosine hydroxylasepositive neurons was seen. When cultured with GDNF MASCs differentiatedinto GABAergic and dopaminergic neurons. When cultured with exclusivedifferentiation into GABAergic neurons was seen after three weeks.

FIG. 15. Undifferentiated MASCs were implanted around a parietal infarctcaused by ligation of middle cerebral artery in the brain of Wistarrats. Rats were maintained on cyclosporin and function of the paralyzedlimbs examined 6 weeks after injection of the MASCs. As control, animalsreceived saline injections or media conditioned by MASCs. Results areshown for limb placement testing 6 weeks after transplantation of theMASCs or control solutions. Functional improvement to levels equivalentto that of sham animals was only seen in rats transplanted with MASCS.

FIG. 16 Undifferentiated MASCs were implanted around a parietal infarctcaused by ligation of middle cerebral artery in the brain of Wistarrats. Rats were maintained on cyclosporin and function of the paralyzedlimbs examined 6 weeks after injection of the MASCs. After 2 and 6weeks, animals were sacrificed to determine neural phenotype. Because ofautofluorescence of the brain following transplantation with eGFP⁺cells, we had to resort to immunohistochemical analysis of the graft.The majority of eGFP⁺ cells were detected in the grafted area itself at2 weeks. After 6 weeks, eGFP⁺ cells migrated outside the graft. At 2weeks, cells staining with an anti-eGFP antibody remained spherical innature and ranged from 10-30 μm in diameter. After 6 weeks, eGFP⁺ cellswere significantly smaller and neurites could be seen in the graftedarea, extending out to the normal brain tissue. Presence of human cellswas confirmed by staining with a human specific nuclear antibody, NuMa(not shown). This antibody will in the future be used to identify humancells in the graft allowing double and triple staining withimmunofluorescent antibodies. Using human specific anti-nestinantibodies, we detected small clusters of nestin-positive cells in thesame location of the graft as the NuMa-positive cells and GFP⁺ cells,suggestive of neuroectodermal differentiation. In addition, we foundpositive staining for β-tubulin III and Neurofilament-68 and -160, OligoMarker and GFAP, suggesting differentiation to neuronal and glial cells.

FIG. 17 shows immunohistochemical and Western blot analysis forcytokeratin 18 and 19 after MASCs were treated with HGF and KGF. After14 days, large epithelioid cells could be seen that expressedcytokeratin 18 and 19.

DETAILED DESCRIPTION OF THE INVENTION

Whether stem cells that are committed to a certain lineage have theability of undergoing a genetical re-programming similar to what occursin the “cloning process” or “trans-differentiate” is not known. Thepresent inventors have shown that multipotent stem cells persist evenafter birth in multiple organs (such as marrow, liver, brain) whenpurified from these organs and cultured in vitro can proliferate withoutobvious senescence and can differentiate into multiple cell types,different from the tissues they were derived from. The phenotype of stemcells derived from different organs with “plasticity” is similar(CD45⁻CD44⁻HLA-DR⁻HLA-calss I⁻oct3/4 mRNA⁺ and hTRT⁺). In addition, thecharacteristics of such stem cells are similar to that of, for instance,primordial germ cells from which they may be a direct descendant.

The present inventors have evidence that a small fraction of marrowcells, as well as cells in brain and liver, express genes commonly onlyfound in ES or EG cells (oct-4, Rex-1). Furthermore, the presentinventors have detected eGFP+ cells in marrow and brain of newborn micetransgenic for the oct-4/eGFP construct, further demonstrating thatoct-4 expressing cells are present in tissues other than germ cells inpost-embryonic life. Therefore, a small number of stem cells may persistthroughout an adult, living in different organs that have multipotentcharacteristics. This explains the perceived plasticity of stem cellsderived from multiple organs.

Selection and Phenotype of Multipotent Adult Stem Cells

The present invention provides multipotent adult stem cells (MASCs),isolated from human or mouse (and other species) adults, newborns, orfetuses, that can differentiate to form bone cells, cartilage,adipocytes, fibroblasts, bone marrow stromal cells, skeletal muscle,smooth muscle, cardiac muscle, endothelium, epithelial cells(keratinocytes), hemopoietic, glial, neuronal and oligodendrocyteprogenitor cells. These cells exhibit differentiation phenotypes moreakin to an embryonic stem cell than to any adult-derived stem celldescribed to date.

The multipotent adult stem cells described herein were isolated by themethod developed by the inventors, who identified a number of specificcell surface markers that characterize the MASCs. The method of thepresent invention can be used to isolate multipotent adult stem cellsfrom any adult, child, or fetus, of human, murine and other speciesorigin. In addition, in mouse, these cells have been isolated from brainand liver. It is therefore now possible for one of skill in the art toobtain bone marrow aspirates, brain or liver biopsies, and possiblyother organs, and isolate the cells using positive or negative selectiontechniques known to those of skill in the art, relying upon the surfacemarkers expressed on these cells, as identified by the inventors,without undue experimentation.

A. MASCs from Human Marrow:

To select the multipotent adult stem cells, bone marrow mononuclearcells are derived from bone marrow aspirates, which can be obtained bystandard means known to those of skill in the art (see, for example,Muschler, G. F., et al., J. Bone Joint Surg. Am. (1997) 79(11):1699-709, Batinic, D., et al., Bone Marrow Transplant. (1990)6(2):103-7). The multipotent adult stem cells are present within thebone marrow (or other organs such as liver or brain) but do not expressthe common leukocyte antigen CD45 or erythroblast specific glycophorin-A(Gly-A). The mixed population of cells is subjected to a Ficoll Hypaqueseparation. Cells are then subjected to negative selection usinganti-CD45 and anti-Gly-A antibodies, depleting the population of CD45⁺and Gly-A⁺ cells, and recovering the remaining approximately 0.1% ofmarrow mononuclear cells. Cells can also be plated in fibronectin coatedwells and cultured as described below for 2-4 weeks after which thecells are depleted of CD45⁺ and Gly-A⁺ cells. Alternatively, positiveselection is used to isolate cells using a combination of cell-specificmarkers identified by the inventors and described herein, such as theleukemia inhibitory factor (LIF) receptor. Both positive and negativeselection techniques are known to those of skill in the art, andnumerous monoclonal and polyclonal antibodies suitable for negativeselection purposes are also known in the art (see, for example,Leukocyte Typing V, Schlossman, et al., Eds. (1995) Oxford UniversityPress) and are commercially available from a number of sources.Techniques for mammalian cell separation from a mixture of cellpopulations have also been described by Schwartz, et al., in U.S. Pat.No. 5,759,793 (magnetic separation), Basch, et al., J. Immunol. Methods(1983) 56: 269 (immunoaffinity chromatography), and Wysocki and Sato,Proc. Natl. Acad. Sci. (USA) (1978) 75: 2844 (fluorescence-activatedcell sorting). (FIG. 1A) Recovered CD45⁻/GlyA⁻ cells are plated ontoculture dishes coated with 5-115 ng/ml (preferably about 7-10 ng/ml)serum fibronectin or other appropriate matrix coating. Cells aremaintained in Dulbecco Minimal Essential Medium (DMEM) or otherappropriate cell culture medium, supplemented with 1-50 ng/ml(preferably about 5-15 ng/ml) platelet-derived growth factor-BB(PDGF-BB), 1-50 ng/ml (preferably about 5-15 ng/ml) epidermal growthfactor (EGF), 1-50 ng/ml (preferably about 5-15 ng/ml) insulin-likegrowth factor (IGF), or 100-10,000 IU (preferably about 1,000 IU) LIF,with 10⁻¹⁰ to 10⁻⁸ M dexamethasone or other appropriate steroid, 2-10μg/ml linoleic acid, and 0.05-0.15 μM ascorbic acid. Other appropriatemedia include, for example, MCDB, MEM, IMDM, and RPMI. Cells can eitherbe maintained without serum, in the presence of 1-2% fetal calf serum,or, for example, in 1-2% human AB serum or autologous serum. (FIG. 1B)

The present inventors have shown that MASCs cultured at low densityexpress the LIF-R, and these cells do not or minimally express CD44whereas cells cultured at high density, that have characteristics ofMSC, loose expression of LIF-R but express CD44. 1-2% CD45⁻GlyA⁻ cellsare CD44⁻ and <0.5% CD45⁻GlyA⁻ cells are LIF-R⁺. FACS selected cellswere subjected to quantitative RT-PCR (real time PCR) for oct-4 mRNA.oct-4 mRNA levels were 5 fold higher in CD45⁻GlyA⁻CD44⁻ and 20-foldhigher in CD45⁻GlyA⁻LIF-R⁺ cells than in unsorted CD45⁻GlyA⁻ cells.Sorted cells were plated in MASC culture with 10 ng/mL EGF, PDGF-BB andLIF. The frequency with which MASC started growing was 30-fold higher inCD45⁻GlyA⁻LIF-R⁺ cells and 3 fold higher in CD45⁻GlyA⁻CD44⁻ cells thanin unsorted CD45⁻GlyA⁻ cells.

When human cells are re-seeded at <0.5×10³ cells/cm², cultures growpoorly and die. When re-seeded at >10×10³ cells/cm² every 3 days, cellsstop proliferating after <30 cell doublings and, as will be discussedbelow, this also causes loss of differentiation potential. Whenre-seeded at 2×10³ cells/cm² every 3 days, >40 cell doublings canroutinely be obtained, and some populations have undergone >70 celldoublings. Cell doubling time was 36-48 h for the initial 20-30 celldoublings. Afterwards cell-doubling time was extended to as much as60-72 h. (FIG. 2)

Telomere length of MASCs from 5 donors (age 2 years-55 years) culturedat reseeding densities of 2×10³ cells/cm² for 23-26 cell doublings wasbetween 11-13 kB. This was 3-5 kB longer than telomere length of bloodlymphocytes obtained from the same donors. Telomere length of cells from2 donors evaluated after 23 and 25 cell doublings resp. and again after35 cells doublings, was unchanged. The karyotype of these MASCS wasnormal. (FIG. 3)

B. MASCs from Murine Tissues:

Marrow from C57/BL6 mice was obtained and mononuclear cells or cellsdepleted of CD45 and GlyA positive cells plated under the same cultureconditions used for human MASCs (10 ng/mL human PDGF-BB and EGF). Whenmarrow mononuclear cells were plated, we depleted CD45⁺ cells 14 daysafter initiation of culture to remove hemopoietic cells. As for humanMASCs, cultures were re-seeded at 2,000 cells/cm² every 2 celldoublings. In contrast to what we saw with human cells, when freshmurine marrow mononuclear cells depleted on day 0 of CD45⁺ cells wereplated in MASCs culture, no growth was seen. When murine marrowmononuclear cells were plated, and cultured cells 14 days later depletedof CD45⁺ cells, cells with the morphology and phenotype similar to thatof human MASCs appeared. When cultured with PDGF-BB and EFG alone, celldoubling was slow (>6 days) and cultures could not be maintained beyond10 cell doublings. Addition of 100-10,000 ng/mL (preferably 1,000 IU)LIF significantly improved cell growth and >70 cell doublings have beenobtained.

Marrow, brain or liver mononuclear cells from 5-day old FVB/N mice wereplated in MASCs cultures with EGF, PDGF-BB and LIF on fibronectin. 14days later, CD45⁺ cells were removed and cells maintained in MASCsculture conditions as described above. Cells with morphology andphenotype similar to that of human MASCs and murine marrow C57/Bl6 MASCsgrew in cultures initiated with marrow, brain or liver cells from FVB/Nmice.

C. Phenotype of MASCs.

1. Human MASCs.

Immunophenotypic analysis by FACS of human MASCs obtained after 22-25cell doublings showed that cells do not express CD31, CD34, CD36, CD38,CD45, CD50, CD62E and -P, HLA-DR, Muc18, STRO-1, cKit, Tie/Tek; andexpress low levels of CD44, HLA-class I, and β2-microglobulin, butexpress CD10, CD13, CD49b, CD49e, CDw90, Flk1 (N>10).

Once cells undergo >40 doublings in cultures re-seeded at 2×10³/cm², thephenotype becomes more homogenous and no cell expressed HLA-class-I orCD44 (n=6). When cells were grown at higher confluence, they expressedhigh levels of Muc18, CD44, HLA-class I and β2-microglobulin, which issimilar to the phenotype described for MSC (N=8) (Pittenger, Science(1999) 284: 143-147).

Immunhistochemistry showed that human MASCs grown at 2×10³/cm² seedingdensity express EGF-R, TGF-R1 and -2, BMP-R1A, PDGF-R1a and -B, and thata small subpopulation (between 1 and 10%) of MASCs stain with anti-SSEA4antibodies (Kannagi R, EMBO J 2:2355-61, 1983).

Using Clontech cDNA arrays we evaluated the expressed gene profile ofhuman MASCs cultured at seeding densities of 2×10³/cm² for 22 and 26cell doublings and found the following profiles:

-   -   A. MASCS do not express CD31, CD36, CD62E, CD62P, CD44-H, cKit,        Tie, receptors for IL1, IL3, IL6, IL11, G-CSF, GM-CSF, Epo,        Flt3-L, or CNTF, and low levels of HLA-class-I, CD44-E and        Muc-18 mRNA.    -   B. MASCs express mRNA for the cytokines BMP1, BMP5, VEGF, HGF,        KGF, MCP1; the cytokine receptors Flk1, EGF-R, PDGF-R1α, gp130,        LIF-R, activin-R1 and -R2, TGFR-2, BMP-R1A; the adhesion        receptors CD49c, CD49d, CD29; and CD10.    -   C. MASCs express mRNA for hTRT and TRF1; the POU-domain        transcription factor oct-4 c sox-2 (required with oct-4 to        maintain undifferentiated state of ES/EC, Uwanogho D, Mech Dev        49:23-36, 1995), sox-11 (neural development), sox-9        (chondrogenesis, Lefebvre V, Matrix Biol 16:529-40, 1998);        homeodeomain transcription factors: Hoxa4 and -a5 (cervical and        thoracic skeleton specification; organogenesis of respiratory        tract, Packer A I, Dev Dyn 17:62-74, 2000), Hox-a9        (myelopoiesis, Lawrence H, Blood 89:1922, 1997), D1x4        (specification of forebrain and peripheral structures of head,        Akimenko M A, J Neurosci 14:3475-86, 1994), MSX1 (embryonic        mesoderm, adult heart and muscle, chondro- and osteogenesis,        Foerst-Potts L, Dev Dyn 209:70-84, 1997), PDX1 (pancreas,        Offield M F, Development 122:983-95, 1996)    -   D. Presence of oct-4, LIF-R, and hTRT mRNA has been confirmed by        RT-PCR.    -   E. In addition RT-PCR showed that Rex-1 mRNA (required with        oct-4 to maintain ES in an undifferentiated state, Rosfjord E,        Biochem Biophys Res Commun 203:1795-802, 1997) and Rox-1 mRNA        (required with oct-4 for transcription of Rex-1, Ben-Shushan E,        Cell Biol 18:1866-78, 1998) are expressed in MASCs.

oct-4 is a transcription factor expressed in the pregastrulation embryo,early cleavage stage embryo, cells of the inner cell mass of theblastocyst, and in embryonic carcinoma (EC) cells (Nichols J, et al Cell95:379-91, 1998), and is down-regulated when cells are induced todifferentiate. Expression of oct-4 plays an important role indetermining early steps in embryogenesis and differentiation. oct-4, incombination with Rox-1, causes transcriptional activation of theZn-finger protein Rex-1, also required for maintaining ESundiffereniated (Rosfjord E, Rizzino A. Biochem Biophys Res Commun203:1795-802, 1997; Ben-Shushan E, et al, Mol Cell Biol 18:1866-78,1998. In addition, sox-2, expressed in ES/EC, but also in other moredifferentiated cells, is needed together with oct-4 to retain theundifferentiated state of ES/EC (Uwanogho D, Rex M, Cartwright E J,Pearl G, Healy C, Scotting P J, Sharpe P T: Embryonic expression of thechicken Sox2, Sox3 and Sox11 genes suggests an interactive role inneuronal development. Mech Dev 49:23-36, 1995). Maintenance of murine EScells and primordial germ cells requires presence of LIF whereas thisrequirement is not so clear for human and non-human primate ES cells.

The present inventors observed that oct-4, Rex-1 and Rox-1 are expressedin MASCs derived from human and murine marrow and from murine liver andbrain. Human MASCs express the LIF-R and stain positive with SSEA-4.Initial experiments show that human MASCs are enriched by selection ofLIF-R⁺ cells even though it is not yet clear if their growth is affectedby LIF. In contrast, LIF aids in the growth of murine MASCs. Finally,oct-4, LIF-R, Rex-1 and Rox-1 mRNA levels increase in human MASCscultures beyond 30 cell doublings, which results in phenotypically morehomogenous cells. In contrast, MASCs cultured at high-density loseexpression of these markers. This is associated with senescence before40 cell doublings and loss of differentiation to cells other thanchondroblasts, osteoblasts and adipocytes. Thus, the presence of oct-4,combined with Rex-1, Rox-1, sox-2, and the LIF-R are markers thatcorrelate with presence of the most primitive cells in MASCs cultures.

The present inventors have examined mice transgenic for an oct-4promoter-eGFP gene. In these animals, eGFP expression is seen inprimordial germ cells as well as in germ cells after birth. As MASCsexpress oct-4, the present inventors tested whether eGFP positive cellscould be found in marrow, brain, and liver of these animals after birth.eGFP⁺ cells (1% brightest population) were sorted from marrow, brain andliver from 5 day-old mice. When evaluated by fluorescence microscopy,<1% of sorted cells from brain and marrow were eGFP⁺. oct-4 mRNA couldbe detected by Q-RT-PCR in the sorted population. Sorted cells have beenplated under conditions that support murine MASCs (fibronectin coatedwells with EGF, PDGF, LIF). Cells survived but did not expand. Whentransferred to murine embryonic fibroblasts, cell growth was seen. Whenreplated again under MASC conditions, cells with morphology andphenotype of MASCs were detected.

2. Murine MASCs.

As for human cells, C57/BL6 MASCs cultured with EGF, PDGF-BB and LIF areCD44 and HLA-class-I negative, stain positive with SSEA-4, and expresstranscripts for oct-4, LIF-R, Rox-1 and sox-2. Likewise, MASCs fromFVB/N marrow, brain and liver express oct3/4 mRNA.

Culturing Multipotent Adult Stem Cells

Multipotent adult stem cells (MASCs) isolated as described herein can becultured using methods of the invention. Briefly, culture in low-serumor serum-free medium is preferred to maintain the cells in theundifferentiated state. Serum-free medium used to culture the cells, asdescribed herein, is supplemented as described in Table I.

TABLE I Insulin 10-50 μg/ml (10 μg/ml)* Transferrin 0-10 μg/ml (5.5μg/ml) Selenium 2-10 ng/ml (5 ng/ml) Bovine serum albumin (BSA) 0.1-5μg/ml (0.5 μg/ml) Linoleic acid 2-10 μg/ml (4.7 μg/ml) Dexamethasone0.005-0.15 μM (.01 μM) L-ascorbic acid 2-phosphate 0.1 mM Low-glucoseDMEM (DMEM-LG) 40-60% (60%) MCDB-201 40-60% (40%) Fetal calf serum 0-2%Platelet-derived growth 5-15 ng/ml (10 ng/ml) Epidermal growth factor5-15 ng/ml (10 ng/ml) Insulin like growth factor 5-15 ng/ml (10 ng/ml)Leukemia inhibitory factor 10-10,000 IU (1,000 IU) *Preferredconcentrations are shown in parentheses.

Because MASCs express the LIF-R and some cells express oct-4, it wastested whether addition of LIF would improve culture. Addition of 10ng/mL LIF to human MASCs did not affect short-term cell growth (samecell doubling time till 25 cell doublings, level of oct-4 expression).In contrast to what was seen with human cells, when fresh murine marrowmononuclear cells depleted on day 0 of CD45⁺ cells were plated in MASCsculture, no growth was seen. When murine marrow mononuclear cells wereplated, and cultured cells 14 days later depleted of CD45⁺ cells, cellswith the morphology and phenotype similar to that of human MASCsappeared. This suggests that factors secreted by hemopoietic cells maybe needed to support initial growth of murine MASCs. When cultured withPDGF-BB and EFG alone, cell doubling was slow (>6 days) and culturescould not be maintained beyond 10 cell doublings. Addition of 10 ng/mLLIF significantly enhanced cell growth.

Once established in culture, cells can be frozen and stored as frozenstocks, using DMEM with 40% FCS and 10% DMSO. Other methods forpreparing frozen stocks for cultured cells are also known to those ofskill in the art.

Inducing MASCs to Differentiate to Form Committed Progenitors andTissue-Specific Cell Types

Using appropriate growth factors, chemokines, and cytokines, MASCs ofthe present invention can be induced to differentiate to form a numberof cell lineages, including, for example, a variety of cells ofmesodermal origin as well as cell from neuroectodermal origin (glialcells, oligodendrocytes, and neurons) as well as endodermal origin.

A. Splanchnic Mesoderm

1. Osteoblasts: Confluent MASCs were cultured with about 10⁻⁶-10⁻⁸M(preferably about 10⁻⁷M) dexamethasone, β-glycerophosphate and 5-20 mM(preferably 10 mM) ascorbic acid. To demonstrate presence ofosteoblasts, we used Von Kossa staining (silver reduction of CaPo4), orantibodies against bone sialoprotein, osteonectin, osteopontin andosteocalcin (immunohistochemistry/Western). After 14-21 days ofculture, >80% of cells stained positive with these antibodies. (FIGS. 5,6)

2. Chondroblasts: MASCs were trypsinized, and cultured in serum-freeDMEM supplemented with 50-1.00 ng/mL (preferably 100 ng/mL) TGF-β1 inmicromass suspension culture. Small aggregates of cartilage formed inthe bottom of the tubes that stained positive with toluidin blue.Collagen type I was detected initially throughout the micromass (day 5)but after 14 days was only detected in the outer layer of fibrillouscartilage. Collagen type II became detectable after 5 days and stronglystained the micromass by day 14. Staining for bone sialoprotein wasnegative or minimally positive in the outer fibrillous cartilage layer.Variable staining was found for osteonectin, osteocalcin andosteopontin. Presence of collagen type II was confirmed by Western blotand RT-PCR. In addition, RT-PCR on cells recovered after 5 days showedpresence of the cartilage specific transcription factors CART1 andCD-RAP1. (FIGS. 5, 6)

3. Adipocyte: To induce adipocyte differentiation, about 10⁻⁷ to about10⁻⁶ M (preferably about 10⁻⁷ M) dexamethasone, about 50 to about 200μg/ml (preferably about 100 μg/ml) insulin or media supplemented withapproximately 20% horse serum can be used. Adipocyte differentiation canbe detected by examination with light microscopy, staining with oil-red,or detection of lipoprotein lipase (LPL), adipocyte lipid-bindingprotein (aP2), or peroxisome proliferator-activated receptor gamma(PPAR). Methods for detection of cellular markers and products are knownto those of skill in the art, and can include detection using specificligands, such as, for example, troglitazone (TRO) and rosiglitazone(RSG), which bind to PPARγ. (FIGS. 5, 6)

4. Expressed gene profile of cartilage and bone. The present inventorsexamined genes expressed upon differentiation to osteoblasts andchondroblasts. In particular, they examined the expressed gene profileof MASCs (n=3) and MASCs switched to osteoblast or chondroblast cultureconditions for two days to determine whether a relative homogenousswitch to the two specific lineages is seen, using Clonetech andInvitrogen cDNA arrays. A partial list of changes detected is shown intable 2. This is by no means a conclusive evaluation of the expressedgene profile in MASCs, osteoblasts and chondroblasts. However, theresults indicate that differentiation of MASCs to bone and cartilageinduces significant and divergent changes in expressed gene profile,consistent with the observation that most cells within a culture can beinduced to differentiate along a given pathway.

TABLE 2 differentially expressed genes in MASCS, osteoblasts andchondroblasts Loss Acquisition/increase Family Osteoblast orchondroblast osteoblast chondroblast Transcription factors oct-4, sox-2,Hoxa4, 5, 9; Dlx4, Hox7, hox11, sox22 Sox-9, FREAC, hox-11, hox7, PDX1,hTRT, TRF1 CART1, Notch3 Cell cycle Cyclins, cdk's Cdki's Cdki'sAdhesion receptors and ECM syndecan-4; dystroglycan syndecan-4, decorin,lumican, collagen-II, fibronectin, decorin, integrin α2, α3, β1,fibronectin, bone sialoprotein, cartilage glycoprotein, cartilageTIMP-1, CD44, β8, β5 integrin oligomeric matrix protein, MMPs and TIMPs,N-cadherin, CD44, α1 and α6 integrin Cytokine-R/cytokines FLK1, LIF-R,RAR-α, PTHr-P, Leptin-R, VitD3-R, VitD3-R, BMP2, BMP7 RARγ, EGF-R,PDGF-R1a and FGF-R3, FGF-R2, Estrogen-R, -B, TGF-R1 and -2, BMP-R1A,wnt-7a, VEGF-C, BMP2 BMP1 and 4, HGF, KGF, MCP1

5. Expressed gene profile of bone by subtractive hybridization: Thepresent inventors used a subtraction approach to identify geneticdifferences between undifferentiated MASCs and committed progeny. Poly-AmRNA was extracted from undifferentiated MASCs and cells induced todifferentiate to the osteoblast lineage for 2 days. Subtraction andamplification of the differentially expressed cDNAs was done using thePCR-Select kit from Clonetech, as per manufacturer's recommendationwithout modification. We started to analyze gene sequences expressed inday 2 osteoblast cultures but not in undifferentiated MASCs.

-   1) The present inventors sequenced 86 differentially expressed    cDNA-sequences. We confirmed by Northern that the mRNAs are indeed    specifically expressed in day 2 osteoblast progenitors and not    MASCs. The sequences were compared (using the BLAST algorithm) to    the following databases: SwissProt, GenBank protein and nucleotide    collections, ESTs, murine and human EST contigs.-   2) Sequences were categorized by homology: 8 are transcription    factors, 20 are involved in cell metabolism; 5 in chromatin repair;    4 in the apoptosis pathway; 8 in mitochondrial function; 14 are    adhesion receptors/ECM components; 19 are published EST sequences    with unknown function and 8 are novel.-   3) For 2 of the novel sequences, the present inventors started to    perform Q-RT-PCR on MASCs induced to differentiate to bone for 12 h,    24 h, 2 d, 4 d, 7 d and 14 d from 3 individual donors. Genes are    expressed during the initial 2 and 4 days of differentiation    respectively, and down regulated afterwards.-   4) The present inventors have also started to analyze genes present    in undifferentiated MASCs but not day 2 osteoblasts. Thirty    differentially expressed genes have been sequenced and 5 of them are    EST sequences or unknown sequences.

B. Muscle

Differentiation to any muscle phenotype required that MASCs be allowedto become confluent prior to induction of differentiation.

1. Skeletal muscle: To induce skeletal muscle cell differentiation,confluent MASCs cells were treated with about 1 to about 3 μM(preferably about 3 μM) 5-azacytidine in MASC expansion medium for 24hours. Cultures were then maintained in MASCs medium. Differentiationwas evaluated by Western blot and immunohistochemistry. Skeletal muscledifferentiation in vitro can be demonstrated by detecting sequentialactivation of Myf-5, Myo-D, Myf-6, myogenin, desmin, skeletal actin andskeletal myosin, either by immunohistochemistry or Western blot analysisusing standard techniques known to those of skill in the art andcommercially available antibodies. Five days after induction with either5-azacytidine the Myf5, Myo-D and Myf6 transcription factors could bedetected in approximately 50% of cells. After 14-18 days, Myo-D wasexpressed at significantly lower levels, whereas Myf5 and Myf6persisted. Desmin and skeletal actin were detected as early as four daysafter induction, and skeletal myosin at 14 days. Byimmunohistochemistry, 70-80% of cells expressed mature muscle proteinsafter 14 days. Treatment with 5-azacitidine resulted in expression ofGata4 and Gata6 during the first week of culture. In addition, lowlevels of troponin-T could be detected from day two onwards. Smoothmuscle actin was detected at two days after induction and persisted for14 days. When 20% horse serum was added, a fusion of myoblasts intomyotubes that were multinucleated was seen. (FIG. 7) Using doublefluorescent labeling we could show that transduced myoblasts could becaused to fuse with differentially lateral myocytes (FIG. 8).

2. Smooth muscle: Smooth muscle cells can also be induced by culturingMASCs in serum-free medium, without growth factors, supplemented withhigh concentrations (about 50 to about 200 ng/ml, preferably about 100ng/ml) of platelet-derived growth factor (PDGF). Cells should preferablybe confluent at initiation of differentiation. Terminally differentiatedsmooth muscle cells can be identified by detecting expression of desmin,smooth muscle specific actin, and smooth muscle specific myosin bystandard methods known to those of skill in the art. Smooth muscle actinwas detected from day two onwards and smooth muscle myosin after 14days. Approximately 70% of cells stained positive with anti-smoothmuscle actin and myosin antibodies. A presence of myogenin was seen fromday four onwards and desmin after 6 days. Myf5 and Myf6 proteins werealso detected after 2-4 days, which persisted till day 15. No Myo-D wasdetected. (FIG. 7)

3. Cardiac muscle: Cardiac muscle differentiation can be accomplished byadding about 5 to about 200 ng/ml (preferably about 100 ng/ml) basicfibroblast growth factor (bFGF) to the standard serum-free culture mediawithout growth factors, as previously described. Confluent MASCs wereexposed to μM (preferably about 3 μM) 5-azacytidine and to 10⁻⁵-10⁻⁷ M(preferably 10⁻⁶ M) retinoic acid, and then cultured in MASC expansionmedium afterwards. Alternatively, MASCs were cultured with either ofthese inducers alone or a combination of both and then cultured inserum-free medium with 50-200 ng/mL (preferably 100 ng/mL FGF2 or acombination of 5-20 ng/mL (preferably 10 ng/mL) BMP-4 and 100 ng/mLFGF2. We found expression of proteins consistent with cardiomyocytes.Gata4 and Gata6 were expressed as early as day 2 and persisted till day15. Cardiac troponin-T was expressed after day 4 and cardiac troponin-Ifrom day 6 on, while we could detect ANP after day 11. These cardiacproteins were detected in >70% of cells by immuno-histochemistry on day15. We found the transcription factor Myf6 from day 2 on. Expression ofdesmin started on day 6 and myogenin on day 2. We also found skeletalactin. When the cultures were maintained for >3 weeks, cells formedsyncithia. We also saw infrequent spontaneous contractions occurring inthe cultures, which were propagated over several mm distance. (FIG. 7)

C. Endothelial Cells MASCs express Flk1 but not CD34, PECAM, E- andP-selectin, CD36, Tie/Tek or Flt1. When MASCs were cultured serum-freeMASCs medium with 20 ng/mL VEGF we saw the appearance of CD34 on thecell surface and cells expressed vWF by day 14 (immuno-fluorescence)(FIGS. 9, 10). In addition, cells expressed Tie, Tek, Flk1 and Flt1,PECAM, P-selectin and E-selectin, and CD36. Results from thehistochemical staining were confirmed by Western blot. When VEGF inducedcells were cultured on matrigel or collagen type IV, vascular tubeformation was seen. (FIGS. 9, 10)

D. Hemopoietic Cells As MASCs differentiate into CD34⁺ endothelial cellsand recent studies show that CD34⁻Flk1⁺ cells can be induced todifferentiate into endothelial cells as well as hemopoietic cells, wetested whether MASCs could be induced to differentiate in hemopoieticprecursors. MASCs were replated on collagen type IV in PDGF-BB- andEGF-containing MASCs medium with 5% FCS and 100 ng/mL SCF that wasconditioned by the AFT024 feeder, a fetal liver derived mesenchymal linethat supports murine and human repopulating stem cells ex vivo. Cellsrecovered from these cultures expressed cKit, cMyb, Gata2 and G-CSF-Rbut not CD34 (RT-PCR). Because hemopoiesis is induced by factors thatare released by embryonal visceral endoderm, we co-cultured human MASCswith βGal⁺ murine EBs in the presence of human SCF, Flt3-L, Tpo and Epo.In 2 separate studies, we detected a small population of βGal⁻ cellsthat expressed human CD45.

E. Stromal cells: The inventors induced “stromal” differentiation byincubating MASCs with IL-1α, FCS, and horse serum. To demonstrate thatthese cells can support hemopoiesis, feeders were irradiated at 2,000cGy and CD34⁺ cord blood cells plated in contact with the feeder. After1, 2 and 5 weeks, progeny was replated in methylcellulose assay todetermine the number of colony forming cells (CFC). A 3-5-fold expansionof CFC was seen after 2 weeks and maintenance of CFC at 5 weeks, whichwas similar to cultures of CD34⁺ cells in contact with the murine fetalliver derived feeder, AFT024.

F. Neuronal Cells Surprisingly, MASCs induced with VEGF, the hemopoieticcytokines SCF, Flt3-L, Tpo and Epo in MASCs medium containing EGFconditioned by the hematopoietic supportive feeder AFT024 differentiatedinto glial-fibrilar-acidic-protein (GFAP) positive astrocytes,galactocerebroside (GalC) positive oligodendrocytes and neurofilamentpositive neurons (FIG. 11) The inventors hypothesized that production ofFGF2 by the AFT024 feeders and addition of EGF to the cultures mightinduce differentiation to neuronal cells in vitro.

They then examined the effect FGF2, known to play a key role in neuraldevelopment and ex vivo culture of neural precursors, on MASCs derivedneural development. When <50% confluent cultures of human marrow derivedMASCs (n=7) that had been cultured with EGF and PDGF-BB were switched tomedium containing 50-500 ng/mL (preferably 100 ng/mL) FGF2,differentiation to cells expressing of astrocytes, oligodendrocytes andneurons was seen after 2-4 weeks (FIG. 11) After two weeks in culture,26±4% of cells were GFAP positive, 28±3% GalC positive and 46±5%neurofilament-200 positive. When reexamined after three weeks, fewercells were GFAP or GalC positive, but 20±2% cells stained positive forβ-tubulin-III, 22±3% for neurofilament-68, 50±3% for neurofilament-160,20±2% for neurofilament-200, 82±5% for neuron-specific-enolase (NSE) and80±2% for microtubule-associated-protein-2 (MAP2) (FIG. 11) GABA,parvalbumin, tyrosine hydroxylase, DOPA-decarboxylase, and tryptophanhydroxylase were not detected. The number of neurites per neuronincreased from 3±1, to 5±1 and 7±2 from 2, 3 to 4 weeks afterdifferentiation. Differentiation to cells with characteristics ofastrocytes, oligodendrocytes and neurons was confirmed by demonstratingpresence of GFAP, myelin basic protein (MBP) and neurofilament-200 byWestern blot and RT-PCR analysis in FGF2 treated but not MASCs).

FGF-9, first isolated from a glioblastoma cell line, inducesproliferation of glial cells in culture. FGF-9 is found in vivo inneurons of the cerebral cortex, hippocampus, substantia nigra, motornuclei of the brainstem and Purkinje cell layer. When cultured for 3weeks with 5-50 ng/mL (preferably 10 ng/mL) FGF-9 and EGF MASCsgenerated astrocytes, oligodendrocytes and GABAergic and dopaminergic.During CNS development, FGF-8, expressed at the mid/hindbrain boundaryand by the rostral forebrain, in combination with Sonic hedgehog,induces differentiation of dopaminergic neurons in midbrain andforebrain. It was found that when MASCs were cultured with 5-50 ng/mL(preferably 10 ng/mL) FGF-8 and EGF for 3 weeks both dopaminergic andGABAergic neurons were produced. FGF-10 is found in the brain in verylow amounts and its expression is restricted to the hippocampus,thalamus, midbrain and brainstem where it is preferentially expressed inneurons but not in glial cells. Culture of MASCs in 5-50 ng/mL(preferably 10 ng/mL) FGF-10 and EGF for three weeks generatedastrocytes and oligodendrocytes, but not neurons. FGF-4 is expressed bythe notochord and is required for the regionalisation of the midbrain.When treated with 5-50 ng/mL (preferably 10 ng/mL) FGF-4 and EGF for 3weeks MASCs differentiated into astrocytes and oligodendrocytes but notneurons.

Other growth factors that are specifically expressed in the brain andthat affect neural development in-vivo and in-vitro include brainderived neurotrophic factor (BDNF), glial derived neurotrophic factor(GDNF) and ciliary neurotrophic factor (CNTF). BDNF is a member of thenerve growth factor family that promotes in vitro differentiation ofNSC, human subependymal cells, and neuronal precursors to neurons andpromotes neurite outgrowth of hippocamal stem cells in vivo. Consistentwith the known function of BDNF to support survival of dopaminergicneurons of the substantia nigra, when MASCs were treated with 5-50 ng/mL(preferably 10 ng/mL) BDNF and EGF exclusive differentiation intotyrosine hydroxylase positive neurons was seen. GDNF is a member of theTGF− superfamily. In early neurogenesis, GDNF is expressed in theanterior neuroectoderm suggesting that it may play a key role inneuronal development. GDNF promotes survival of motor neurons inperipheral nerve and muscle and has neurotrophic and differentiationabilities. It was found that 5-50 ng/mL (preferably 10 ng/mL) GDNFinduced MASCs to differentiate into GABAergic and dopaminergic neurons.CNTF, first isolated from ciliary ganglion, is a member of the gp130family of cytokines. CNTF promotes neuronal survival early indevelopment. In embryonic rat hippocampal cultures CNTF increased thenumbers of GABAergic and cholinergic neurons. In addition, it preventedcell death of GABAergic neurons and promoted GABA uptake. 5-50 ng/mL(preferably 10 ng/mL) CNTF exerted the same GABAergic induction on MASCsas they differentiated exclusively into GABAergic neurons after threeweeks of exposure to CNTF.

The fate of MASCs transplanted into rat brain was also examined. 50,000eGFP⁺ MASCs were transplanted stereotactically around a parietal infarctinduced in Wistar rats, maintained on cyclosporin. Limb-placement wastested six weeks after transplant of saline, MASCs conditioned medium,or MASCs. Functional improvement to levels equivalent to that of shamanimals was only seen in rats transplanted with MASCs (FIG. 15).

After two and six weeks, animals were sacrificed to determine neuralphenotype. Because of autofluorescence of the brain followingtransplantation with eGFP⁺ cells, immunohistochemical analysis of thegraft was performed. The majority of eGFP⁺ cells were detected in thegrafted area itself at two weeks (FIG. 16). After five weeks, eGFP⁺cells migrated outside the graft. At two weeks, cells staining with ananti-eGFP antibody remained spherical in nature and ranged from 10-30 μmin diameter. After six weeks, cells with an anti-eGFP antibody weresignificantly smaller and dendrites could be seen in the grafted area,extending out to the normal brain tissue. Presence of human cells wasconfirmed by staining with a human specific nuclear antibody, NuMa. Thisantibody can be used to identify human cells in the graft allowingdouble and triple staining with immunofluorescent antibodies.

Using human specific anti-nestin antibodies, the present inventorsdetected small clusters of nestin-positive cells in the same location ofthe graft as the NuMa-positive cells and GFP⁺ cells, suggestive ofneuroectodermal differentiation. In addition, they found positivestaining for -tubulin III and Neurofilament-68 and -160, Oligo Markerand GFAP, suggesting differentiation to neuronal and glial cells (notshown).

G. Epithelial Cells The inventors treated confluent MASCs (N=4) with 10ng/mL hepatocyte growth factor (HGF), alone or in combination withkeratinocyte growth factor (KGF). After 14 days, large epithelioid cellscould be seen that expressed the HGF receptor, cytokeratin 8, 18 and 19.Presence of cytokeratin-19 suggests possible differentiation to biliaryepithelium. Changing the matrix from fibronectin to a collagen gel ormatrigel improved generation of cytokeratin-18 expressing cells withmorphology of epithelial cells. (FIG. 17)

Single Cell Origin of Differentiated Lineages.

To address if MASCs are clonal, the inventors have sorted by FACS 1 and10 MFG-eGFP transduced eGFP⁺ cells per well and cultured cells togenerate 10⁸ cells. Transduction was done as follows: MASCs replated 24h earlier were exposed for 6 h on 2 sequential days to MFG-eGFP orMSCV-eGFP packaged in the PG13 cell line and 10 μg/mL protamine. Between40-70% of MASCS were transduced. Expression of eGFP persisted for atleast 3 months ex vivo, and persisted in a large fraction of cellsfollowing differentiation. When a single cell was sorted, no growth wasseen in >1,000 wells. However, when 10 cells were deposited/well, cellgrowth was seen in 3% of wells, extensive expansion to >10⁷ cells wasseen in only 0.3% of wells. These cells were then induced todifferentiate into all mesodermal cell types (osteoblasts,chondroblasts, adipocytes, skeletal and smooth muscle cells, andendothelium). Differentiation was again shown by immunohistochemistryand Western blot. Cells were also subjected to inverse PCR todemonstrate that the sequences in the human DNA flanking the viralinsert were similar. The inventors found that the retroviral gene wasinserted in the same site in the human genome in MASCs and differentiateprogeny in 3 independent clones.

MASC Engraftment

The inventors initiated studies to examine if MASCs engraft and persistin vivo.

-   1. The inventors injected eGFP⁺ MASCs intramuscularly into NOD-SCID    mice. Animals were sacrificed 4 weeks later and muscle examined to    determine if, as has been described for human ES cells, teratomas    develop. In 5/5 animals, no teratomas were seen. eGFP positive cells    were detected.-   2. The inventors infused eGFP⁺ MASCs IV intrauterine in fetal SCID    mice. Animals were evaluated immediately after birth. PCR analysis    demonstrated presence of eGFP⁺ cells in heart, lung, liver, spleen    and marrow.-   3. The inventors transplanted MASCs stereotaxically in the intact    brain or infarcted brain of rats, they acquire a phenotype    compatible with neural cells, and persist for at least 6 weeks.

Applications of MASCs

1. osteoblasts: MASCs of the present invention that have been induced todifferentiate to form bone cells can be used as cell therapy or fortissue regeneration in osteoporosis, Paget's disease, bone fracture,osteomyelitis, osteonecrosis, achondroplasia, osteogenesis imperfecta,hereditary multiple exostosis, multiple epiphyseal dysplasia, Marfan'ssyndrome, mucopolysaccharidosis, neurofibromatosis or scoliosis,reconstructive surgery for localized malformations, spina bifida,hemivertebrae or fused vertebrae, limb anomalies, reconstruction oftumor-damaged tissue, and reconstruction after infection, such as middleear infection.

2. chondrocytes: MASCs of the present invention can be induced todifferentiate to form cartilage cells for cell therapy or tissueregeneration in age-related diseases or injuries, in sports-relatedinjuries, or in specific diseases such as rheumatoid arthritis,psoriasis arthritis, Reiter's arthritis, ulcerative colitis, Crohns'disease, ankylosing spondylitis, osteoathritis, reconstructive surgeryof the outer ear, reconstructive surgery of the nose, and reconstructivesurgery of the cricoid cartilage.

3. adipocytes: Adipocytes derived from the MASCs can be used inresculpting during reconstructive or cosmetic surgery, as well as forthe treatment of Type II diabetes. In reconstructive surgery, adipocytesdifferentiated as described by the method of the present invention canbe used for breast reconstruction after mastectomy, for example, or forreshaping tissue lost as a result of other surgery, such as tumorremoval from the face or hand. In cosmetic surgery, adipocytes producedfrom the cells of the present invention by the method of the presentinvention can be used in a variety of techniques, such as breastaugmentation, or for reduction of wrinkles in aging skin. Adipocytesthus derived can also provide an effective in vitro model system for thestudy of fat regulation.

4. fibroblasts: Fibroblasts derived from the MASCs can be used for celltherapy or tissue repair to promote wound healing or to provideconnective tissue support, such as scaffolding for cosmetic surgery.

5. Skeletal muscle: MASCs can be induced to differentiate to formskeletal muscle cells for cell therapy or tissue repair in the treatmentof Duchene muscular dystrophy, Becker muscular dystrophy, myotonicdystrophy, skeletal myopathy, and reconstructive surgery to repairskeletal muscle damage.

6. Smooth muscle: MASCs can be induced to differentiate to form smoothmuscle cells for cell therapy or tissue repair in the treatment ofdevelopmental abnormalities of the gastrointestinal system, such asoesophageal atresia, intestinal atresia, and intussusception, as well asfor replacement of tissues after surgery for bowel infarction orcolocolostomy.

Smooth muscle cells formed from the MASCs of the present invention canalso be used for bladder or uterine reconstruction, forneovascularization, for repair of vessels damaged by, for example,atherosclerosis or aneurysm. Smooth muscle precursor cells (mesangialcells) can be used as an in vitro model for glomerular diseases or forcell therapy or tissue regeneration in diabetic neuropathy. Smoothmuscle precursors can also be used to repair macula densa of the distalconvoluted tubule or juxtaglomerular tissues, which play a role in bloodpressure regulation.

7. cardiomyocytes: Cardiomyocytes derived from the MASCs can be usefulfor cell therapy or tissue repair for treating heart tissue damagedfollowing myocardial infarction, in conjunction with congestive heartfailure, during valve replacement, by congenital heart anomalies, orresulting from cardiomyopathies or endocarditis. Cells can be deliveredlocally, especially by injection, for increased effectiveness.Microglial cells differentiated from MASCs can be used to treat spinalcord injuries and neurodegenerative disorders, such as Huntingtonsdisease, Parkinsons disease, Multiple Sclerosis, and Alzheimers disease,as well as repair of tissues damaged during infectious disease affectingthe central nervous system. Microglial cells that have been geneticallyaltered to produce cytokines can also be used for transplantation forthe treatment of infectious disease in the central nervous system whereaccess is limited due to the blood-brain barrier. Glial cells can alsobe used to produce growth factors or growth factor inhibitors forregeneration of nerve tissue after stroke, as a consequence of multiplesclerosis, amylotropic lateral sclerosis, and brain cancer, as well asfor regeneration after spinal cord injury.

8. stromal cells: Stromal cells derived from the MASCs of the presentinvention can be used as transplant cells for post-chemotherapy bonemarrow replacement, as well as for bone marrow transplantation. Inbreast cancer, for example, a bone marrow aspirate is obtained from apatient prior to an aggressive chemotherapy regimen. Such chemotherapyis damaging to tissues, particularly to bone marrow. MASCs isolated fromthe patient's bone marrow can be expanded in culture to provide enoughautologous cells for re-population of the bone marrow cells. Becausethese cells can differentiate to multiple tissues types, cellsintroduced either locally or systemically provide an added advantage bymigrating to other damaged tissues, where cellular factors in the tissueenvironment induce the cells to differentiate and multiply.

9. endothelial cells: MASCs can be differentiated by the methodsdescribed to produce endothelial cells, which can be used in thetreatment of Factor VIII deficiency, as well as to produce angiogenesisfor neovascularization. Endothelial cells can also provide an in vitromodel for tumor suppression using angiogenic inhibitors, as well as anin vitro model for vasculitis, hypersensitivity and coagulationdisorders. Using these cultured endothelial cells and rapid screeningmethods known to those of skill in the art, thousands of potentiallyuseful therapeutic compounds can be screened in a more timely andcost-effective manner.

10. hematopoietic cells: MASCs can differentiate into hematopoieticcells. Cells of the present invention can therefore be used torepopulate the bone marrow after high dose chemotherapy. Prior tochemotherapy, a bone marrow aspirate is obtained from the patient. Stemcells are isolated by the method of the present invention, and are grownin culture and induced to differentiate. A mixture of differentiated andundifferentiated cells is then reintroduced into the patient's bonemarrow space. Clinical trials are currently underway using hematopoieticstem cells for this purpose. The stem cells of the present invention,however, provide the additional benefit of further differentiation toform cells that can replace those damaged by chemotherapy in othertissues as well as in bone marrow. Hematopoietic cells derived from theMASCs can be further differentiated to form blood cells to be stored inblood banks, alleviating the problem of a limited supply of blood fortransfusions.

11. Neuroectodermal cells: Microglial cells differentiated from MASCscan be used to treat spinal cord injuries and neurodegenerativedisorders, such as Huntingtons disease, Parkinsons disease, MultipleSclerosis, and Alzheimers disease, as well as repair of tissues damagedduring infectious disease affecting the central nervous system.Microglial cells that have been genetically altered to produce cytokinescan also be used for transplantation for the treatment of infectiousdisease in the central nervous system where access is limited due to theblood-brain barrier. Glial cells can also be used to produce growthfactors or growth factor inhibitors for regeneration of nerve tissueafter stroke, as a consequence of multiple sclerosis, amylotropiclateral sclerosis, and brain cancer, as well as for regeneration afterspinal cord injury. MASCs induced to form oligodendrocytes andastrocytes, for example, can be used for transplant into demyelinatedtissues, especially spinal cord, where they function to myelinate thesurrounding nervous tissues. This technique has been demonstratedeffective in mice, using embryonic stem cells as the source ofoligodendrocyte and astrocyte precursors (Brustle, O., et al., Science(1999) 285: 754-756). The MASCs of the present invention exhibit thebroad range of differentiation characteristic of embryonic cells, butprovide the added advantage of contributing autologous cells fortransplant.

The cells of the present invention can be used in cell replacementtherapy and/or gene therapy to treat congenital neurodegenerativedisorders or storage disorders such as, for instance,mucopolysaccharidosis, leukodystrophies (globoid-cell leukodystrophy,Canavan disease), fucosidosis, GM2 gangliosidosis, Niemann-Pick,Sanfilippo syndrome, Wolman disease, and Tay Sacks. They can also beused for traumatic disorders such as stroke, CNS bleeding, and CNStrauma; for peripheral nervous system disorders such as spinal cordinjury or syringomyelia; for retinal disorders such as retinaldetachment, macular degeneration and other degenerative retinaldisorders, and diabetic retinopathy.

12. Ectodermal epithelial cells: Moreover, the epithelial cells of thepresent invention can also be used in cell replacement therapy and/orgene therapy to treat or alleviate symptoms of skin disorders such asalopecia, skin defects such as burn wounds, and albinism.

13. Endodermal epithelial cells: Epithelial cells derived from the MASCof the present invention can be used in cell replacement therapy and/orgene therapy to treat or alleviate symptoms of several organ diseases.The cells could be used to treat or alleviate congenital liverdisorders, for example, storage disorders such as mucopolysaccharidosis,leukodystrophies, GM2 gangliosidosis; increased bilirubin disorders, forinstance Crigler-Najjar syndrome; ammonia disorders such as inbornerrors of the urea-cycle, for instance Ornithine decarboxylasedeficiency, citrullinemia, and argininosuccinic aciduria; inborn errorsof amino acids and organic acids such as phenylketoinuria, hereditarytyrosinemia, and Alpha1-antitrypsin deficiency; and coagulationdisorders such as factor VIII and IX deficiency. The cells can also beused to treat acquired liver disorders due to viral infections. Thecells of the present invention can also be used in ex vivo applicationssuch as to generate an artificial liver (akin to kidney dialysis), toproduce coagulation factors and to produce proteins or enzymes generatedby liver epithelium.

These epithelial cells of the present invention can also be used in cellreplacement therapy and/or gene therapy to treat or alleviate symptomsof biliary disorders such as biliary cirthosis and biliary atresia.

The epithelial cells of the present invention can also be used in cellreplacement therapy and/or gene therapy to treat or alleviate symptomsof pancreas disorders such as pancreatic atresia, pancreas inflammation,and Alpha1-antitrypsin deficiency. Further, as pancreas epithelium canbe made from the cells of the present invention, and as neural cells canbe made, beta-cells can be generated. These cells can be used for thetherapy of diabetes (subcutaneous implantation or intra-pancreas orintra-liver implantation. Further, the epithelial cells of the presentinvention can also be used in cell replacement therapy and/or genetherapy to treat or alleviate symptoms of gut epithelium disorders suchas gut atresia, inflammatory bowel disorders, bowel infarcts, and bowelresection.

14. Modification of MASC to ensure absence of senescence under less thanoptimal culture conditions: Although MASC have long telomeres (12 kb)and the telomere length is not different in cells from donors ofdifferent ages. Upon ex vivo culture of the MASC, telomeres do notshorten for an extended period of time, i.e., for over 4 months in exvivo culture (or >35 cell doublings). This may persist longer.Telomerase is present in MASC derived from people of all ages. When MASCcells are cultured under confluent conditions, senescence occurs andtelomers begin to shorten. As extensive expansion in relative high densecultures may be preferable for production, commercial or other purposes,MASC can be transduced/transfected with a telomerase-containingconstruct, which will prevent senescence of cells. As these cells couldthen be used for in vivo transplantation, it would be preferable thattelomerase be removed from the cell prior to transplantation. This canbe accomplished by engineering the telomerase construct such that it islocated between two LoxP sites. The Cre recombinase will be able to thenexcize telomerase. Cre can be transfected/transduced into the targetcell using a second vector/plasmid or as part of thetelomerase-containing construct. Cre can be introduced in aconstitutively active form, or as an inducible enzyme, for instance byflanking the protein with one or more mutated ligand binding domains ofthe human estrogen receptor (ER) that can be induced by4-hydroxy-tamoxifen (OHT), but not natural ER ligands, or by using atetracyclin or rapamacine inducible, or other drug inducible system.

15. Approaches for transplantation to prevent immune rejection:

a. universal donor cells: MASC can be manipulated to serve as universaldonor cells for cell and gene therapy to remedy genetic or otherdiseases and to replace enzymes. Although undifferentiated MASC expressno HLA-type I, HLA-type II antigens or beta-2 microglobulin, somedifferentiated progeny express at least type I HLA-antigens. MACS can bemodified to serve as universal donor cells by eliminating HLA-type I andHLA-type II antigens, and potentially introducing the HLA-antigens fromthe prospective recipient to avoid that the cells become easy targetsfor NK-mediated killing, or become susceptible to unlimited viralreplication and/or malignant transformation. Elimination of HLA-antigenscan be accomplished by homologous recombination or via introduction ofpoint-mutations in the promoter region or by introduction of apointmutation in the initial exon of the antigen to introduce astop-codon, such as with chimeroplasts. Transfer of the host HLA-antigencan be achieved by retroviral, lentiviral, adeno associated virus orother viral transduction or by transfection of the target cells with theHLA-antigen cDNA's. MASC can be used to establish and set amount or agiven range or level of a protein in the body or blood.

b. Intrauterine transplant to circumvent immune recognition: MASC can beused in intrauterine transplantation setting to correct geneticabnormalities, or to introduce cells that will be tolerated by the hostprior to immune system development. This can be a way to make humancells in large quantities such as blood, in animals or it could be usedas a way to correct human embryo genetic defects by transplanting cellsthat make the correct protein or enzyme.

16. Gene therapy: Until now, human cells used for gene therapy have beenessentially limited to bone marrow and skin cells, because other typesof cells could not be extracted from the body, grown in culture,genetically altered, then successfully reimplanted into the patient fromwhom the tissue was taken. (Anderson, W. F., Nature (1998) 392: 30;Anderson, W. F., Scientific American (1995) 273: 1-5; Anderson, W. F.Science (1992) 256: 808-813) MASCs of the present invention can beextracted and isolated from the body, grown in culture in theundifferentiated state or induced to differentiate in culture, andgenetically altered using a variety of techniques, especially viraltransduction. Uptake and expression of genetic material is demonstrable,and expression of foreign DNA is stable throughout development.Retroviral and other vectors for inserting foreign DNA into stem cellsare known to those of skill in the art. (Mochizuki, H., et al., J. Virol(1998) 72(11): 8873-8883; Robbins, P., et al., J. Virol. (1997) 71(12):9466-9474; Bierhuizen, M., et al., Blood (1997) 90(9): 3304-3315;Douglas, J., et al., Hum. Gene Ther. (1999) 10(6): 935-945; Zhang, G.,et al., Biochem. Biophys. Res. Commun. (1996) 227(3): 707-711). Oncetransduced using a retroviral vector, enhanced green fluorescent protein(eGFP) expression persists in terminally differentiated muscle cells,endothelium, and c-Kit positive cells derived from the isolated MASCs,demonstrating that expression of retroviral vectors introduced into MASCpersists throughout differentiation. Terminal differentiation wasinduced from cultures initiated with 10 eGFP⁺ cells previouslytransduced by retroviral vector and sorted a few weeks into the initialMASC culture period.

Hematopoietic stem cells, although limited in differentiation potential,demonstrate utility for gene therapy (see Kohn, D. B., Curr. Opin.Pediatr. (1995) 7: 56-63). The cells of the present invention provide awider range of differentiated cell types which can retain transduced ortransfected DNA when terminally differentiated, as demonstrated by thefact that terminally differentiated muscle cells, endothelium, and c-Kitpositive cells retained enhanced green fluorescent protein expressionalthough the retroviral vector had been introduced into theundifferentiated stem cell.

MASCs of the present invention provide other advantages overhematopoietic stem cells for gene therapy, as well. Stem cells of thepresent invention are relatively easy to isolate from bone marrowaspirates obtained under local anesthesia, easy to expand in culture,and easy to transfect with exogenous genes. Adequate numbers ofhematopoietic stem cells for the same purpose must be isolated from atleast one liter of marrow and the cells are difficult to expand inculture (see Prockop, D. J., Science (1997) 276: 71-74).

Candidate genes for gene therapy include, for example, genes encodingApolipoprotein E (which has been correlated with risk for Alzheimer'sdisease and cardiovascular disease), MTHFR (variants of which have beenassociated with increased homocysteine levels and risk of stroke),Factor V (which has been correlated with risk of thrombosis), ACE(variants of which have been correlated with risk of heart disease),CKR-5 (which has been associated with resistance to HIV), HPRT(hypoxanthine-guanine phosphoribosyl transferase, the absence of whichresults in Lesch-Nyhan disease), PNP (purine nucleoside phosphorylase,the absence of which results in severe immunodeficiency disease), ADA(adenosine deaminase, the absence of which results in severe combinedimmunodeficiency disease), p21 (which has been proposed as a candidategene for treatment for ataxia telangiectasia), p47 (the absence of whichis correlated with lack of oxidase activity in neutrophils of patientswith chronic granulomatous disease, GenBank accession number M55067 andM38755), Rb (the retinoblastoma susceptibility gene associated withtumor formation, GenBank accession number M15400), KVLQT1 (a potassiumchannel protein, with aberrant forms associated with cardiacarrhythmias, GenBank accession number U40990), the dystrophin gene(associated with Duchenne muscular dystrophy, GenBank accession numbersM18533, M17154, and M18026), CFTR (the transmembrane conductanceregulator associated with cystic fibrosis, GenBank accession numberM28668), phosphatidylinositol 3-kinase (associated with ataxiatelangiectasia, GenBank accession number U26455), and VHL (loss ormutation of the protein is associated with Von-Hippel Lindau disease:Latif, F., et al., Science (1993) 260: 1317-1320). Other diseases whichcan be treated effectively using these genetically-altered cellsinclude, Factor IX deficiency, adenosine deaminase deficiency(associated with severe combined immunodeficiency disease, or SCIDS),and diabetes, and deficiencies in glucocerebrosidase, α-iduronidase.

These novel genes can be driven by an inducible promoter so that levelsof enzyme can be regulated. These inducible promoter systems may includea mutated ligand binding domain of the human estrogen receptor (ER)attached to the protein to be produced. This would require that theindividual ingests tamoxifen to allow expression of the protein.Alternatives are tetracyclin on or off systems, RU486, and a rapamycininducible system. An additional method to obtain relative selectiveexpression is to use tissue specific promoters. For instance in thebrain, one can introduce a transgene driven by the neuron-specificenolase promoter (Ad-NSE) or the glial fibrillary acidic proteinpromoter (GFAP) promoter, which will allow almost exclusive expressionin brain tissue. Likewise, endothelial expression only may be obtainedby using the Tec promoter or the VE-cadherin promoter.

Genetically altered MASCs can be introduced locally or infusedsystemically. Human stem cells with more limited differentiationpotential, when transfected with a gene for factor IX, secrete theprotein for at least 8 weeks after systemic infusion into SCID mice.(Keating, A., et al., Blood (1996) 88: 3921.) MASCs of the presentinvention, having a broader differentiation potential than anynon-embryonic stem cell described thus far, provide an added advantagefor systemic or local administration, because they can migrate to avariety of tissues, where cytokines, growth factors, and other factorsinduce differentiation of the cell. The differentiated cell, now a partof the surrounding tissue, retains its ability to produce the proteinproduct of the introduced gene.

In Parkinson's disease, for example, clinical trials have shown thatmesencephalic dopamine neurons obtained from human embryo cadavers cansurvive and function in the brains of patients with Parkinson's disease.PET scans have indicated that [¹⁸F]fluorodopa uptake in the area aroundthe cell graft is increased after transplantation, and remains so for atleast six years in some patients. (See Dunnett, S. and A. Bjorklund,Nature (1999) 399 (Suppl.) A32-A-39; Lindvall, O., Nature Biotech.(1999) 17: 635-636; Wagner, J., et al., Nature Biotech. (1999) 17:653-659.) Unlike the embryonic cells, isolated MASCs as described by thepresent invention provide a ready supply of cells for transplant, yetmaintain the differentiation potential that makes embryonic celltransplant therapy an attractive alternative for disease treatment.

For AIDS therapy, MASCs of the present invention can be geneticallyengineered to produce Rev M10, a transdominant negative mutant of Revthat blocks the function of a wild-type Rev produced in HIV-infectedcells. (Bevec, D. et al., Proc. Natl. Acad. Sci. USA (1992) 89:9870-9874; Ranga, U., et al., Proc. Natl. Acad. Sci. USA (1998) 95(3):1201-1206.) Once induced to differentiate into hematopoietic lineagecells and introduced into the patient, MASCs repopulate the HIV-infectedpatient's depleted T cell supply. Since the genetically altered cellspossess the mutant Rev M10, they will be resistant to the lethal effectsof infection by most strains of HIV.

Genetically altered MASCs can also be encapsulated in an inert carrierto allow the cells to be protected from the host immune system whileproducing the secreted protein. Techniques for microencapsulation ofcells are known to those of skill in the art (see, for example, Chang,P., et al., Trends in Biotech. (1999) 17(2): 78-83). Materials formicroencapsulation of cells include, for example, polymer capsules,alginate-poly-L-lysine-alginate microcapsules, barium poly-L-lysinealginate capsules, barium alginate capsules,polyacrylonitrile/polyvinylchloride (PAN/PVC) hollow fibers, andpolyethersulfone (PES) hollow fibers. U.S. Pat. No. 5,639,275 (Baetge,E., et al.), for example, describes improved devices and methods forlong-term, stable expression of a biologically active molecule using abiocompatible capsule containing genetically engineered cells. Suchbiocompatible immunoisolatory capsules, in combination with the MASCs ofthe present invention, provide a method for treating a number ofphysiologic disorders, including, for example, diabetes and Parkinson'sdisease.

In the diabetic patient, for example, heterologous stem cells which havebeen genetically altered to produce insulin at physiologicallytherapeutic levels can be encapsulated for delivery within the patient'stissues. Alternately, autologous stem cells can be derived from thepatient's own bone marrow aspirate for transduction with a retroviralvector as previously described. Once genetically altered to producephysiologically therapeutic levels of insulin, these cells can beencapsulated as described by Chang or Baetge and introduced into thepatient's tissues where they remain to produce insulin for extendedperiods of time.

Another advantage of microencapsulation of cells of the presentinvention is the opportunity to incorporate into the microcapsule avariety of cells, each producing a biologically therapeutic molecule.MASCs of the present invention can be induced to differentiate intomultiple distinct lineages, each of which can be genetically altered toproduce therapeutically effective levels of biologically activemolecules. MASCs carrying different genetic elements can be encapsulatedtogether to produce a variety of biologically active molecules.

MASCs of the present invention can be genetically altered ex vivo,eliminating one of the most significant barriers for gene therapy. Forexample, a subject's bone marrow aspirate is obtained, and from theaspirate MASCs are isolated. The MASCs are then genetically altered toexpress one or more desired gene products. The MASCs can then bescreened or selected ex vivo to identify those cells which have beensuccessfully altered, and these cells can be reintroduced into thesubject, either locally or systemically. Alternately, MASCs can begenetically altered and cultured to induce differentiation to form aspecific cell lineage for transplant. In either case, the transplantedMASCs provide a stably-transfected source of cells that can express adesired gene product. Especially where the patient's own bone marrowaspirate is the source of the MASCs, this method provides animmunologically safe method for producing transplant cells. The methodcan be used for treatment of diabetes, cardiac myopathy,neurodegenerative disease, and adenosine deaminase deficiency, to nameonly a few of a multitude of examples. In diabetes, for example, MASCscan be isolated, genetically altered to produce insulin, thentransplanted into the patient suffering from the disease. Where thedisease is associated with autoimmunity, MASCs can be geneticallyaltered to express either an altered MHC or no MHC in order to avoidimmune surveillance. Suppression of MHC expression in transplantedpancreatic islet cells has been successfully performed using anadenoviral vector expressing the E3 region of the viral genome. Cells ofthe present invention can be stably transfected or transduced, as theinventors have demonstrated, and can therefore provide a more permanentsource of insulin for transplant into a diabetic patient.

Donor MASCs, particularly if genetically altered to alter MHCexpression, and autologous MASCs, if genetically altered to express thedesired hemoglobin gene products, can be especially effective in celltherapy for the treatment of sickle cell anemia and thalassemia.

Methods for Genetically Altering MASCs

Cells isolated by the method described herein can be geneticallymodified by introducing DNA or RNA into the cell by a variety of methodsknown to those of skill in the art. These methods are generally groupedinto four major categories: (1) viral transfer, including the use of DNAor RNA viral vectors, such as retroviruses (including lentiviruses),Simian virus 40 (SV40), adenovirus, Sindbis virus, and bovinepapillomavirus for example; (2) chemical transfer, including calciumphosphate transfection and DEAE dextran transfection methods; (3)membrane fusion transfer, using DNA-loaded membranous vesicles such asliposomes, red blood cell ghosts, and protoplasts, for example; and (4)physical transfer techniques, such as microinjection, electroporation,or direct “naked” DNA transfer. MASCs can be genetically altered byinsertion of pre-selected isolated DNA, by substitution of a segment ofthe cellular genome with pre-selected isolated DNA, or by deletion of orinactivation of at least a portion of the cellular genome of the cell.Deletion or inactivation of at least a portion of the cellular genomecan be accomplished by a variety of means, including but not limited togenetic recombination, by antisense technology (which can include theuse of peptide nucleic acids, or PNAs), or by ribozyme technology, forexample. Insertion of one or more pre-selected DNA sequences can beaccomplished by homologous recombination or by viral integration intothe host cell genome. The desired gene sequence can also be incorporatedinto the cell, particularly into its nucleus, using a plasmid expressionvector and a nuclear localization sequence. Methods for directingpolynucleotides to the nucleus have been described in the art. Thegenetic material can be introduced using promoters that will allow forthe gene of interest to be positively or negatively induced usingcertain chemicals/drugs, to be eliminated following administration of agiven drug/chemical, or can be tagged to allow induction by chemicals(including but not limited to the tamoxifen responsive mutated estrogenreceptor) expression in specific cell compartments (including but notlimited to the cell membrane).

Homologous Recombination

Calcium phosphate transfection, which relies on precipitates of plasmidDNA/calcium ions, can be used to introduce plasmid DNA containing atarget gene or polynucleotide into isolated or cultured MASCs. Briefly,plasmid DNA is mixed into a solution of calcium chloride, then added toa solution which has been phosphate-buffered. Once a precipitate hasformed, the solution is added directly to cultured cells. Treatment withDMSO or glycerol can be used to improve transfection efficiency, andlevels of stable transfectants can be improved usingbis-hydroxyethylamino ethanesulfonate (BES). Calcium phosphatetransfection systems are commercially available (e.g., ProFection® fromPromega Corp., Madison, Wis.).

DEAE-dextran transfection, which is also known to those of skill in theart, may be preferred over calcium phosphate transfection wheretransient transfection is desired, as it is often more efficient.

Since the cells of the present invention are isolated cells,microinjection can be particularly effective for transferring geneticmaterial into the cells. Briefly, cells are placed onto the stage of alight microscope. With the aid of the magnification provided by themicroscope, a glass micropipette is guided into the nucleus to injectDNA or RNA. This method is advantageous because it provides delivery ofthe desired genetic material directly to the nucleus, avoiding bothcytoplasmic and lysosomal degradation of the injected polynucleotide.This technique has been used effectively to accomplish germlinemodification in transgenic animals.

Cells of the present invention can also be genetically modified usingelectroporation. The target DNA or RNA is added to a suspension ofcultured cells. The DNA/RNA-cell suspension is placed between twoelectrodes and subjected to an electrical pulse, causing a transientpermeability in the cell's outer membrane that is manifested by theappearance of pores across the membrane. The target polynucleotideenters the cell through the open pores in the membrane, and when theelectric field is discontinued, the pores close in approximately one to30 minutes.

Liposomal delivery of DNA or RNA to genetically modify the cells can beperformed using cationic liposomes, which form a stable complex with thepolynucleotide. For stabilization of the liposome complex, dioleoylphosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC)can be added. A recommended reagent for liposomal transfer isLipofectin® (Life Technologies, Inc.), which is commercially available.Lipofectin®, for example, is a mixture of the cationic lipidN-[1-(2,3-dioleyloyx)propyl]-N-N-N-trimethyl ammonia chloride and DOPE.Delivery of linear DNA, plasmid DNA, or RNA can be accomplished eitherin vitro or in vivo using liposomal delivery, which may be a preferredmethod due to the fact that liposomes can carry larger pieces of DNA,can generally protect the polynucleotide from degradation, and can betargeted to specific cells or tissues. A number of other deliverysystems relying on liposomal technologies are also commerciallyavailable, including Effectene™ (Qiagen), DOTAP (Roche MolecularBiochemicals), FuGene 6™ (Roche Molecular Biochemicals), andTransfectam® (Promega). Cationic lipid-mediated gene transfer efficiencycan be enhanced by incorporating purified viral or cellular envelopecomponents, such as the purified G glycoprotein of the vesicularstomatitis virus envelope (VSV-G), in the method of Abe, A., et al. (J.Virol. (1998) 72: 6159-6163).

Gene transfer techniques which have been shown effective for delivery ofDNA into primary and established mammalian cell lines usinglipopolyamine-coated DNA can be used to introduce target DNA into MASCs.This technique is generally described by Loeffler, J. and Behr, J.,Methods in Enzymology (1993) 217: 599-618.

Naked plasmid DNA can be injected directly into a tissue mass formed ofdifferentiated cells from the isolated MASCs. This technique has beenshown to be effective in transferring plasmid DNA to skeletal muscletissue, where expression in mouse skeletal muscle has been observed formore than 19 months following a single intramuscular injection. Morerapidly dividing cells take up naked plasmid DNA more efficiently.Therefore, it is advantageous to stimulate cell division prior totreatment with plasmid DNA.

Microprojectile gene transfer can also be used to transfer genes intoMASCs either in vitro or in vivo. The basic procedure formicroprojectile gene transfer was described by J. Wolff in GeneTherapeutics (1994) at page 195. Briefly, plasmid DNA encoding a targetgene is coated onto microbeads, usually 1-3 micron sized gold ortungsten particles. The coated particles are placed onto a carrier sheetinserted above a discharge chamber. Once discharged, the carrier sheetis accelerated toward a retaining screen. The retaining screen forms abarrier which stops further movement of the carrier sheet while allowingthe polynucleotide-coated particles to be propelled, usually by a heliumstream, toward a target surface, such as a tissue mass formed ofdifferentiated MASCs. Microparticle injection techniques have beendescribed previously, and methods are known to those of skill in the art(see Johnston, S. A., et al., Genet. Eng. (NY) (1993) 15: 225-236;Williams, R. S., et al., Proc. Natl. Acad. Sci. USA (1991) 88:2726-2730; Yang, N. S., et al., Proc. Natl. Acad. Sci. USA (1990) 87:9568-9572).

Signal peptides can be attached to plasmid DNA, as described bySebestyen, et al. (Nature Biotech. (1998) 16: 80-85), to direct the DNAto the nucleus for more efficient expression.

Viral vectors are used to genetically alter MASCs of the presentinvention and their progeny. Viral vectors are used, as are the physicalmethods previously described, to deliver one or more target genes,polynucleotides, antisense molecules, or ribozyme sequences, forexample, into the cells. Viral vectors and methods for using them todeliver DNA to cells are well known to those of skill in the art.Examples of viral vectors which can be used to genetically alter thecells of the present invention include, but are not limited to,adenoviral vectors, adeno-associated viral vectors, retroviral vectors(including lentiviral vectors), alphaviral vectors (e.g., Sindbisvectors), and herpes virus vectors.

Retroviral vectors are effective for transducing rapidly-dividing cells,although a number of retroviral vectors have been developed toeffectively transfer DNA into non-dividing cells as well (Mochizuki, H.,et al., J. Virol. (1998) 72: 8873-8883). Packaging cell lines forretroviral vectors are known to those of skill in the art. Packagingcell lines provide the viral proteins needed for capsid production andvirion maturation of the viral vector. Generally, these include the gag,pol, and env retroviral genes. An appropriate packaging cell line ischosen from among the known cell lines to produce a retroviral vectorwhich is ecotropic, xenotropic, or amphotropic, providing a degree ofspecificity for retroviral vector systems.

A retroviral DNA vector is generally used with the packaging cell lineto produce the desired target sequence/vector combination within thecells. Briefly, a retroviral DNA vector is a plasmid DNA which containstwo retroviral LTRs positioned about a multicloning site and SV40promoter so that a first LTR is located 5 to the SV40 promoter, which isoperationally linked to the target gene sequence cloned into themulticloning site, followed by a 3 second LTR. Once formed, theretroviral DNA vector can be transferred into the packaging cell lineusing calcium phosphate-mediated transfection, as previously described.Following approximately 48 hours of virus production, the viral vector,now containing the target gene sequence, is harvested.

Targeting of retroviral vectors to specific cell types was demonstratedby Martin, F., et al., (J. Virol. (1999) 73: 6923-6929), who usedsingle-chain variable fragment antibody directed against the surfaceglycoprotein high-molecular-weight melanoma-associated antigen fused tothe amphotropic murine leukemia virus envelope to target the vector todelivery the target gene to melanoma cells. Where targeted delivery isdesired, as, for example, when differentiated cells are the desiredobjects for genetic alteration, retroviral vectors fused to antibodyfragments directed to the specific markers expressed by each celllineage differentiated from the MASCs of the present invention can beused to target delivery to those cells.

Lentiviral vectors are also used to genetically alter cells of theinvention. Many such vectors have been described in the literature andare known to those of skill in the art. Salmons, B. and Gunzburg, W. H.,“Targeting of Retroviral Vectors for Gene Therapy,” Hum. Gene Therapy(1993) 4: 129-141. These vectors have been effective for geneticallyaltering human hematopoietic stem cells (Sutton, R., et al., J. Virol.(1998) 72: 5781-5788). Packaging cell lines have been described forlentivirus vectors (see Kafri, T., et al., J. Virol. (1999) 73: 576-584;Dull, T., et al., J. Virol. (1998) 72: 8463-8471).

Recombinant herpes viruses, such as herpes simplex virus type I (HSV-1)have been used successfully to target DNA delivery to cells expressingthe erythropoietin receptor (Laquerre, S., et al., J. Virol. (1998) 72:9683-9697). These vectors can also be used to genetically alter thecells of the present invention, which the inventors have demonstrated tobe stably transduced by a viral vector.

Adenoviral vectors have high transduction efficiency, can incorporateDNA inserts up to 8 Kb, and can infect both replicating anddifferentiated cells. A number of adenoviral vectors have been describedin the literature and are known to those of skill in the art (see, forexample, Davidson, B. L., et al., Nature Genetics (1993) 3: 219-223;Wagner, E., et al., Proc. Natl. Acad. Sci. USA (1992)89: 6099-6103).Methods for inserting target DNA into an adenovirus vector are known tothose of skill in the art of gene therapy, as are methods for usingrecombinant adenoviral vectors to introduce target DNA into specificcell types (see Wold, W., Adenovirus Methods and Protocols, HumanaMethods in Molecular Medicine (1998), Blackwell Science, Ltd.). Bindingaffinity for certain cell types has been demonstrated by modification ofthe viral vector fiber sequence. Adenovirus vector systems have beendescribed which permit regulated protein expression in gene transfer(Molin, M., et al., J. Virol. (1998) 72: 8358-8361). A system has alsobeen described for propagating adenoviral vectors with geneticallymodified receptor specificities to provide transductional targeting tospecific cell types (Douglas, J., et al., Nature Biotech. (1999) 17:470-475). Recently described ovine adenovirus vectors even address thepotential for interference with successful gene transfer by preexistinghumoral immunity (Hofmann, C., et al., J. Virol. (1999) 73: 6930-6936).

Adenovirus vectors are also available which provide targeted genetransfer and stable gene expression using molecular conjugate vectors,constructed by condensing plasmid DNA containing the target gene withpolylysine, with the polylysine linked to a replication-incompetentadenovirus. (Schwarzenberger, P., et al., J. Virol. (1997) 71:8563-8571.)

Alphavirus vectors, particularly the Sindbis virus vectors, are alsoavailable for transducing the cells of the present invention. Thesevectors are commercially available (Invitrogen, Carlsbad, Calif.) andhave been described in, for example, U.S. Pat. No. 5,843,723, as well asby Xiong, C., et al., Science (1989) 243: 1188-1191; Bredenbeek, P. J.,et al., J. Virol. (1993) 67: 6439-6446; and Frolov, I., et al., Proc.Natl. Acad. Sci. USA (1996) 93: 11371-11377.

The inventors have shown that MASC possess good transduction potentialusing the eGFP-MND lentiviral vector described by Robbins, et al. (J.Virol. (1997) 71(12): 9466-9474) and eGFP-MGF vector. Using this method,30-50% of MASC can be transduced after a short exposure of 4.6 hours toan enhanced green fluorescent protein (eGFP) vector containingsupernatants made in PA3-17 packaging cells (an amphotropic packagingcell line derived from NIH 3T3 fibroblasts and described by Miller, A.D., and C. Buttimore in Mol. Cell. Biol. (1986) 6: 2895-2902), combinedwith protamine (8 mg/ml). Expression of eGFP persists throughout theculture of undifferentiated MASC. In addition, transfection usinglipofectamine has been successfully used to introduce transgenes inMAPCs.

Successful transfection or transduction of target cells can bedemonstrated using genetic markers, in a technique that is known tothose of skill in the art. The green fluorescent protein of Aequoreavictoria, for example, has been shown to be an effective marker foridentifying and tracking genetically modified hematopoietic cells(Persons, D., et al., Nature Medicine (1998) 4: 1201-1205). Alternativeselectable markers include the β-Gal gene, the truncated nerve growthfactor receptor, drug selectable markers (including but not limited toNEO, MTX, hygromycin)

17. MASCs Are Useful For Tissue Repair: The stem cells of the presentinvention can also be used for tissue repair. The inventors havedemonstrated that MASCs of the present invention differentiate to form anumber of cell types, including fibroblasts, osteoblasts, chondrocytes,adipocytes, skeletal muscle, endothelium, stromal cells, smooth muscle,cardiac muscle, and hemopoietic cells. For example, MASCs induced todifferentiate into osteoblasts, by the method previously describedherein, can be implanted into bone to enhance the repair process, toreinforce weakened bone, or to resurface joints. MASCs induced todifferentiate into chondrocytes, by the method previously described, canbe injected into joints to resurface joint cartilage. Caplan, et al.(U.S. Pat. No. 5,855,619) describe a biomatrix implant including acontracted gel matrix into which mesenchymal stem cells have beenincorporated. The implant is designed for repair of a tissue defect,especially for injury to tendon, ligament, meniscus, or muscle.Cartilage, for example, can be formed by the addition of chondrocytes inthe immediate area around a porous, 3-dimensional scaffold made, forexample, of collagen, synthetic polyglycolic acid fibers, or syntheticpolylactic fibers. The inventors have shown that MASCs of the presentinvention differentiate to form chondrocytes, for example, which can bedeposited in and around a collagen, synthetic polyglycolic, or syntheticpolylactic or other scaffold material to provide an implant tofacilitate tissue repair.

Matrices are also used to deliver cells of the present invention tospecific anatomic sites, where particular growth factors incorporatedinto the matrix, or encoded on plasmids incorporated into the matrix foruptake by the cells, can be used to direct the growth of the initialcell population. DNA can be incorporated within pores of the matrix, forexample, during the foaming process used in the formation of certainpolymer matrices. As the polymer used in the foaming process expands, itentraps the DNA within the pores, allowing controlled and sustainedrelease of plasmid DNA. Such a method of matrix preparation is describedby Shea, et al., in Nature Biotechnology (1999) 17: 551-554.

Plasmid DNA encoding cytokines, growth factors, or hormones can betrapped within a polymer gene-activated matrix carrier, as described byBonadio, J., et al., Nature Medicine (1999) 5: 753-759. Thebiodegradable polymer is then implanted near a broken bone, for example,where MASCs are implanted and take up the DNA, which causes the MASCs toproduce a high local concentration of the cytokine, growth factor, orhormone, accelerating healing of the damaged tissue.

Cells provided by the present invention, or MASCs isolated by the methodof the present invention, can be used to produce tissues or organs fortransplantation. Oberpenning, et al. (Nature Biotechnology (1999) 17:149-155) reported the formation of a working bladder by culturing musclecells from the exterior canine bladder and lining cells from theinterior of the canine bladder, preparing sheets of tissue from thesecultures, and coating a small polymer sphere with muscle cells on theoutside and lining cells on the inside. The sphere was then insertedinto a dog's urinary system, where it began to function as a bladder.Nicklason, et al., Science (1999) 284: 489-493, reported the productionof lengths of vascular graft material from cultured smooth muscle andendothelial cells. Other methods for forming tissue layers from culturedcells are known to those of skill in the art (see, for example, Vacanti,et al., U.S. Pat. No. 5,855,610). These methods can be especiallyeffective when used in combination with cells of the present invention,which have a broader range of differentiation than anypreviously-described non-embryonic stem cells.

MASCs of the present invention can be used to repopulate heart musclecells by either direct injection into the area of tissue damage or bysystemic injection, allowing the cells to home to the cardiac tissues.This method can be particularly effective if combined with angiogenesis.Both the methods of injection and methods for promoting angiogenesis areknown to those of skill in the art. The MASCs of the present inventionprovide a broader differentiation range to provide a more varied sourceof cells for cardiac or other tissue repair utilizing these techniques.

MASCs of the present invention are also useful, for example, for thepurpose of repopulating the bone marrow after high dose chemotherapy.Prior to chemotherapy, a bone marrow aspirate is obtained from thepatient. Stem cells are isolated by the method of the present invention,and are grown in culture and induced to differentiate. A mixture ofdifferentiated and undifferentiated cells is then reintroduced into thepatient's bone marrow space. Clinical trials are currently underwayusing hematopoietic stem cells for this purpose. The MASCs of thepresent invention, however, provide the additional benefit of furtherdifferentiation to form cells that can replace those damaged bychemotherapy in other tissues as well as in bone marrow.

Alternately, the method described by Lawman, et al. (WO 98/42838) can beused to change the histocompatibility antigen of stem cells from anallogeneic donor or donors. Using this method, panels of available bonemarrow transplants can be generated for preparation of frozen stocks,storage, and administration to patients who are unable, as in leukemiapatients, for example, to provide their own bone marrow forreconstitution.

Re-population of a patient's immune system cells or blood cells can beaccomplished, for example, by isolating autologous stem cells from thepatient, culturing those cells to expand the population, thenreintroducing the cells into the patient. This method can beparticularly effective where the immune system or bone marrow cells mustbe depleted by radiation and/or chemotherapy for therapeutic purposes,such as in the case, for example, of patients diagnosed with multiplemyeloma, non-Hodgkins lymphoma, autoimmune disease, or solid tumorcancers.

For the treatment of leukemias, autoimmune disease, or genetic diseasessuch as sickle cell anemia or thalassemia, re-population of thepatient's blood or immune system cells with allogeneic cells of thepresent invention, or isolated by the method of the present invention,can be performed, particularly when the histocompatibility antigen hasbeen altered in the manner described by Lawman, et al. (WO 98/42838).

For the purposes described herein, either autologous or allogeneic MASCsof the present invention can be administered to a patient, either indifferentiated or undifferentiated form, genetically altered orunaltered, by direct injection to a tissue site, systemically, on oraround the surface of an acceptable matrix, or in combination with apharmaceutically acceptable carrier.

19. MASCs Provide a Model System for Studying Differentiation Pathways:Cells of the present invention are useful for further research intodevelopmental processes, as well. Ruley, et al. (WO 98/40468), forexample, have described vectors and methods for inhibiting expression ofspecific genes, as well as obtaining the DNA sequences of thoseinhibited genes. Cells of the present invention can be treated with thevectors such as those described by Ruley, which inhibit the expressionof genes that can be identified by DNA sequence analysis. The cells canthen be induced to differentiate and the effects of the alteredgenotype/phenotype can be characterized.

Hahn, et al. (Nature (1999) 400: 464-468) demonstrated, for example,that normal human epithelial fibroblast cells can be induced to undergotumorigenic conversion when a combination of genes, previouslycorrelated with cancer, were introduced into the cells.

Control of gene expression using vectors containing inducible expressionelements provides a method for studying the effects of certain geneproducts upon cell differentiation. Inducible expression systems areknown to those of skill in the art. One such system is theecdysone-inducible system described by No, D., et al. Proc. Natl. Acad.Sci. USA (1996) 93: 3346-3351.

MASCs can be used to study the effects of specific genetic alterations,toxic substances, chemotherapeutic agents, or other agents on thedevelopmental pathways. Tissue culture techniques known to those ofskill in the art allow mass culture of hundreds of thousands of cellsamples from different individuals, providing an opportunity to performrapid screening of compounds suspected to be, for example, teratogenicor mutagenic.

For studying developmental pathways, MASCs can be treated with specificgrowth factors, cytokines, or other agents, including suspectedteratogenic chemicals. MASCs can also be genetically modified usingmethods and vectors previously described. Furthermore, MASCs can bealtered using antisense technology or treatment with proteins introducedinto the cell to alter expression of native gene sequences. Signalpeptide sequences, for example, can be used to introduce desiredpeptides or polypeptides into the cells. A particularly effectivetechnique for introducing polypeptides and proteins into the cell hasbeen described by Rojas, et al., in Nature Biotechnology (1998) 16:370-375. This method produces a polypeptide or protein product that canbe introduced into the culture media and translocated across the cellmembrane to the interior of the cell. Any number of proteins can be usedin this manner to determine the effect of the target protein upon thedifferentiation of the cell. Alternately, the technique described byPhelan et al. (Nature Biotech. (1998) 16: 440-443) can be used to linkthe herpes virus protein VP22 to a functional protein for import intothe cell.

Cells of the present invention can also be genetically engineered, bythe introduction of foreign DNA or by silencing or excising genomic DNA,to produce differentiated cells with a defective phenotype in order totest the effectiveness of potential chemotherapeutic agents or genetherapy vectors.

20. MASCs Provide a Variety of Differentiated and UndifferentiatedCultured Cell Types for High-Throughput Screening: MASCs of the presentinvention can be cultured in, for example, 96-well or other multi-wellculture plates to provide a system for high-throughput screening of, forexample, target cytokines, chemokines, growth factors, or pharmaceuticalcompositions in pharmacogenomics or pharmacogenetics. The MASCs of thepresent invention provide a unique system in which cells can bedifferentiated to form specific cell lineages from the same individual.Unlike most primary cultures, these cells can be maintained in cultureand can be studied over time. Multiple cultures of cells from the sameindividual and from different individuals can be treated with the factorof interest to determine whether differences exist in the effect of thecellular factor on certain types of differentiated cells with the samegenetic makeup or on similar types of cells from genetically differentindividuals. Cytokines, chemokines, pharmaceutical compositions andgrowth factors, for example, can therefore be screened in a timely andcost-effective manner to more clearly elucidate their effects. Cellsisolated from a large population of individuals and characterized interms of presence or absence of genetic polymorphisms, particularlysingle nucleotide polymorphisms, can be stored in cell culture banks foruse in a variety of screening techniques. For example, multipotent adultstem cells from a statistically significant population of individuals,which can be determined according to methods known to those of skill inthe art, provide an ideal system for high-throughput screening toidentify polymorphisms associated with increased positive or negativeresponse to a range of substances such as, for example, pharmaceuticalcompositions, vaccine preparations, cytotoxic chemicals, mutagens,cytokines, chemokines, growth factors, hormones, inhibitory compounds,chemotherapeutic agents, and a host of other compounds or factors.Information obtained from such studies has broad implication for thetreatment of infectious disease, cancer, and a number of metabolicdiseases.

In the method of using MASCs to characterize cellular responses tobiologic or pharmacologic agents, or combinatorial libraries of suchagents, MASCs are isolated from a statistically significant populationof individuals, culture expanded, and contacted with one or morebiologic or pharmacologic agents. MASCs can be induced to differentiate,where differentiated cells are the desired target for a certain biologicor pharmacologic agent, either prior to or after culture expansion. Bycomparing the one or more cellular responses of the MASC cultures fromindividuals in the statistically significant population, the effects ofthe biologic or pharmacologic agent can be determined. Alternately,genetically identical MASCs, or cells differentiated therefrom, can beused to screen separate compounds, such as compounds of a combinatoriallibrary. Gene expression systems for use in combination with cell-basedhigh-throughput screening have been described (see Jayawickreme, C. andKost, T., Curr. Opin. Biotechnol. (1997) 8: 629-634). A high volumescreening technique used to identify inhibitors of endothelial cellactivation has been described by Rice, et al., which utilizes a cellculture system for primary human umbilical vein endothelial cells.(Rice, et al., Anal. Biochem. (1996) 241: 254-259.) The cells of thepresent invention provide a variety of cell types, both terminallydifferentiated and undifferentiated, for high-throughput screeningtechniques used to identify a multitude of target biologic orpharmacologic agents. Most important, the cells of the present inventionprovide a source of cultured cells from a variety of genetically diverseindividuals who may respond differently to biologic and pharmacologicagents.

MASCs can be provided as frozen stocks, alone or in combination withprepackaged medium and supplements for their culture, and can beadditionally provided in combination with separately packaged effectiveconcentrations of appropriate factors to induce differentiation tospecific cell types. Alternately, MASCs can be provided as frozenstocks, prepared by methods known to those of skill in the art,containing cells induced to differentiate by the methods describedhereinabove.

21. MASCs and Genetic Profiling: Genetic variation can have indirect anddirect effects on disease susceptibility. In a direct case, even asingle nucleotide change, resulting in a single nucleotide polymorphism(SNP), can alter the amino acid sequence of a protein and directlycontribute to disease or disease susceptibility. Functional alterationin the resulting protein can often be detected in vitro. For example,certain APO-lipoprotein E genotypes have been associated with onset andprogression of Alzheimer's disease in some individuals.

DNA sequence anomalies can be detected by dynamic-allele specifichybridization, DNA chip technologies, and other techniques known tothose of skill in the art. Protein coding regions have been estimated torepresent only about 3% of the human genome, and it has been estimatedthat there are perhaps 200,000 to 400,000 common SNPs located in codingregions.

Previous investigational designs using SNP-associated genetic analysishave involved obtaining samples for genetic analysis from a large numberof individuals for whom phenotypic characterization can be performed.Unfortunately, genetic correlations obtained in this manner are limitedto identification of specific polymorphisms associated with readilyidentifiable phenotypes, and do not provide further information into theunderlying cause of the disease.

MASCs of the present invention provide the necessary element to bridgethe gap between identification of a genetic element associated with adisease and the ultimate phenotypic expression noted in a personsuffering from the disease. Briefly, MASCs are isolated from astatistically significant population of individuals from whom phenotypicdata can be obtained (see Collins, et al., Genome Research (1998) 8:1229-1231). These MASC samples are then cultured expanded andsubcultures of the cells are stored as frozen stocks, which can be usedto provide cultures for subsequent developmental studies. From theexpanded population of cells, multiple genetic analyses can be performedto identify genetic polymorphisms. For example, single nucleotidepolymorphisms can be identified in a large sample population in arelatively short period of time using current techniques, such as DNAchip technology, known to those of skill in the art (Wang, D., et al.,Science (1998) 280: 1077-1082; Chee, M., et al., Science (1996) 274:610-614; Cargill, M., et al., Nature Genetics (1999) 22: 231-238;Gilles, P., et al., Nature Biotechnology (1999) 17: 365-370; Zhao, L.P., et al., Am. J. Human Genet. (1998) 63: 225-240). Techniques for SNPanalysis have also been described by Syvänen (Syvänen, A., Hum. Mut.(1999) 13: 1-10), Xiong (Xiong, M. and L. Jin, Am. J. Hum. Genet. (1999)64: 629-640), Gu (Gu, Z., et al., Human Mutation (1998) 12: 221-225),Collins (Collins, F., et al., Science (1997) 278: 1580-1581), Howell(Howell, W., et al., Nature Biotechnology (1999) 17: 87-88), Buetow(Buetow, K., et al., Nature Genetics (1999) 21: 323-325), andHoogendoorn (Hoogendoorn, B., et al., Hum. Genet. (1999) 104: 89-93).

When certain polymorphisms are associated with a particular diseasephenotype, cells from individuals identified as carriers of thepolymorphism can be studied for developmental anomalies, using cellsfrom non-carriers as a control. MASCs of the present invention providean experimental system for studying developmental anomalies associatedwith particular genetic disease presentations, particularly, since theycan be induced to differentiate, using certain methods described hereinand certain other methods known to those of skill in the art, to formparticular cell types. For example, where a specific SNP is associatedwith a neurodegenerative disorder, both undifferentiated MASCs and MASCsdifferentiated to form neuronal precursors, glial cells, or other cellsof neural origin, can be used to characterize the cellular effects ofthe polymorphism. Cells exhibiting certain polymorphisms can be followedduring the differentiation process to identify genetic elements whichaffect drug sensitivity, chemokine and cytokine response, response togrowth factors, hormones, and inhibitors, as well as responses tochanges in receptor expression and/or function. This information can beinvaluable in designing treatment methodologies for diseases of geneticorigin or for which there is a genetic predisposition.

In the present method of using MASCs to identify genetic polymorphismsassociated with physiologic abnormalities, MASCs are isolated from astatistically significant population of individuals from whom phenotypicdata can be obtained (a statistically significant population beingdefined by those of skill in the art as a population size sufficient toinclude members with at least one genetic polymorphism) and cultureexpanded to establish MASC cultures. DNA from the cultured cells is thenused to identify genetic polymorphisms in the cultured MASCs from thepopulation, and the cells are induced to differentiate. Aberrantmetabolic processes associated with particular genetic polymorphisms areidentified and characterized by comparing the differentiation patternsexhibited by MASCs having a normal genotype with differentiationpatterns exhibited by MASCs having an identified genetic polymorphism orresponse to putative drugs.

22. MASCs Provide Safer Vaccine Delivery: MASCs cells of the presentinvention can also be used as antigen-presenting cells when geneticallyaltered to produce an antigenic protein. Using multiple alteredautologous or allogeneic progenitor cells, for example, and providingthe progenitor cells of the present invention in combination withplasmids embedded in a biodegradable matrix for extended release totransfect the accompanying cells, an immune response can be elicited toone or multiple antigens, potentially improving the ultimate effect ofthe immune response by sequential release of antigen-presenting cells.It is known in the art that multiple administrations of some antigensover an extended period of time produce a heightened immune responseupon ultimate antigenic challenge. Alternately, MASCs can be used asantigen-presenting cells, in the method of Zhang, et al. (NatureBiotechnology (1998) 1: 1045-1049), to induce T-cell tolerance tospecific antigen.

Many current vaccine preparations incorporate added chemicals and othersubstances, such as antibiotics (to prevent the growth of bacteria invaccine cultures), aluminum (adjuvant), formaldehyde (to inactivatebacterial products for toxoid vaccines), monosodium glutamate(stabilizer), egg protein (component of vaccines prepared usingembryonated chicken eggs), sulfites (stabilizer), and thimerosol (apreservative). Partly due to these added components, there is currentlya broad-based public concern over the safety of vaccine preparations.Thimerosol, for example, contains mercury and is made from a combinationof ethyl mercuric chloride, thiosalicylic acid, sodium hydroxide andethanol. Furthermore, some studies, although inconclusive, havesuggested a possible link between some vaccine components and potentialcomplications such as those diseases commonly associated withautoimmunity. Thus, more effective vaccine therapies are needed andpublic cooperation with vaccine initiatives will be easier to promote ifthere is a greater degree of comfort with the method of vaccination.

MASCs of the present invention can be differentiated to form dendriticcells, which present antigen to T cells and thereby activate them torespond against foreign organisms. These dendritic cells can begenetically altered to express foreign antigens, using techniquespreviously described. A particular advantage of this method of vaccinedelivery lies in the fact that more than one antigen can be presented bya single genetically altered cell.

Differentiated or undifferentiated MASC vaccine vectors of heterologousorigin provide the added advantage of stimulating the immune systemthrough foreign cell-surface markers. Vaccine design experiments haveshown that stimulation of the immune response using multiple antigenscan elicit a heightened immune response to certain individual antigenswithin the vaccine preparation.

Immunologically effective antigens have been identified for hepatitis A,hepatitis B, varicella (chickenpox), polio, diphtheria, pertussis,tetanus, Lyme disease, measles, mumps, rubella, Haemophilus influenzaetype B (Hib), BCG, Japanese encephalitis, yellow fever, and rotavirus,for example.

The method for inducing an immune response to an infectious agent in ahuman subject using MASCs of the present invention can be performed byexpanding a clonal population of multipotent adult stem cells inculture, genetically altering the expanded cells to express one or morepre-selected antigenic molecules to elicit a protective immune responseagainst an infectious agent, and introducing into the subject an amountof genetically altered cells effective to induce the immune response.Methods for administering genetically altered cells are known to thoseof skill in the art. An amount of genetically altered cells effective toinduce an immune response is an amount of cells which producessufficient expression of the desired antigen to produce a measurableantibody response, as determined by methods known to those of skill inthe art. Preferably, the antibody response is a protective antibodyresponse that can be detected by resistance to disease upon challengewith the appropriate infectious agent.

23. MASCs and Cancer Therapy: MASCs of the present invention provide anovel vehicle for cancer therapies. For example, MASCs can be induced todifferentiate to form endothelial cells or precursors which will home toendothelial tissues when delivered either locally or systemically. Thecells participate in formation of blood vessels to supply newly-formedtumors (angiogenesis), and divide and proliferate in the endothelialtissue accordingly. By genetically engineering these cells to undergoapoptosis upon stimulation with an externally-delivered element, thenewly-formed blood vessels can be disrupted and blood flow to the tumorcan be eliminated. An example of an externally-delivered element wouldbe the antibiotic tetracycline, where the cells have been transfected ortransduced with a gene which promotes apoptosis, such as Caspase or BAD,under the control of a tetracycline response element. Tetracyclineresponsive elements have been described in the literature (Gossen, M. &Bujard, H., Proc. Natl. Acad. Sci. USA (1992) 89: 5547-5551), provide invivo transgene expression control in endothelial cells (Sarao, R. &Dumont, D., Transgenic Res. (1998) 7: 421-427), and are commerciallyavailable (CLONETECH Laboratories, Palo Alto, Calif.).

Alternately, undifferentiated MASCs or MASCs differentiated to formtissue-specific cell lineages can be genetically altered to produce aproduct, for export into the extracellular environment, which is toxicto tumor cells or which disrupts angiogenesis (such as pigmentepithelium-derived factor (PEDF), described by Dawson, et al., Science(1999) 285: 245-248). For example, Koivunen, et al., describe cyclicpeptides containing an amino acid sequence which selectively inhibitsMMP-2 and MMP-9 (matrix metalloproteinases associated withtumorigenesis), preventing tumor growth and invasion in animal modelsand specifically targeting angiogenic blood vessels in vivo (Koivunen,E., Nat. Biotech. (1999) 17: 768-774). Where it is desired that cells bedelivered to the tumor site, produce a tumor-inhibitory product, andthen be destroyed, cells can be further genetically altered toincorporate an apoptosis-promoting protein under the control of aninducible promoter.

MASCs also provide a vector for delivery of cancer vaccines, since theycan be isolated from the patient, cultured ex vivo, genetically alteredex vivo to express the appropriate antigens, particularly in combinationwith receptors associated with increased immune response to antigen, andreintroduced into the subject to invoke an immune response to theprotein expressed on tumor cells.

24. Kits Containing MASCs or MASC Isolation and Culture Components:MASCs of the present invention can be provided in kits, with appropriatepackaging material. For example, MASCs can be provided as frozen stocks,accompanied by separately packaged appropriate factors and media, aspreviously described herein, for culture in the undifferentiated state.Additionally, separately packaged factors for induction ofdifferentiation, as previously described, can also be provided.

Kits containing effective amounts of appropriate factors for isolationand culture of a patient's stem cells are also provided by the presentinvention. Upon obtaining a bone marrow aspirate from the patient, theclinical technician only need select the stem cells, using the methoddescribed herein, with the anti-CD45 and anti-glycophorin A provided inthe kit, then culture the cells as described by the method of thepresent invention, using culture medium supplied as a kit component. Thecomposition of the basic culture medium has been previously describedherein.

One aspect of the invention is the preparation of a kit for isolation ofMASCs from a human subject in a clinical setting. Using kit componentspackaged together, MASCs can be isolated from a simple bone marrowaspirate. Using additional kit components including differentiationfactors, culture media, and instructions for inducing differentiation ofMASCs in culture, a clinical technician can produce a population ofantigen-presenting cells (APCs) from the patient's own bone marrowsample. Additional materials in the kit can provide vectors for deliveryof polynucleotides encoding appropriate antigens for expression andpresentation by the differentiated APCs. Plasmids, for example, can besupplied which contain the genetic sequence of, for example, thehepatitis B surface antigen or the protective antigens of hepatitis A,adenovirus, Plasmodium falciparum, or other infectious organisms. Theseplasmids can be introduced into the cultured APCs using, for example,calcium phosphate transfection materials, and directions for use,supplied with the kit. Additional materials can be supplied forinjection of genetically-altered APCs back into the patient, providingan autologous vaccine delivery system.

The invention will be further described by reference to the followingdetailed examples.

EXAMPLES Example 1 Isolation of MASCs from Bone Marrow Mononuclear Cells

Bone marrow mononuclear cells were obtained from bone marrow aspiratesfrom the posterior iliac crest of >80 healthy human volunteers. Ten to100 cubic centimeters of bone marrow was obtained from each subject, asshown in Table 2, which indicates the approximate number of mononuclearcells isolated from each subject. Mononuclear cells (MNC) were obtainedfrom bone marrow by centrifugation over a Ficoll-Paque density gradient(Sigma Chemical Co, St Louis, Mo.). Bone marrow MNC were incubated withCD45 and Glycophorin A microbeads (Miltenyi Biotec, Sunnyvale, Calif.)for 15 minutes and CD45⁺/Gly-A⁺ cells removed by placing the sample infront of a SuperMACS magnet. The eluted cells are 99.5% CD45⁻/GlyA⁻.

As shown in Table 2, depletion of CD45⁺GlyA⁺ cells resulted in recoveryof CD45− GlyA− cells which constituted approximately 0.05 to 0.10% ofthe total bone marrow mononuclear cells.

TABLE 2 Number of Number of MASCs Volume mononuclear BM Number of(estimated by of Bone cells post 45-/GlyA-cell limiting dilution Marrow(cc) ficolled post-MACS assay, LDA) 50 100 millions 100,000 50 25 8060,000 35 25 50 14,000 10 50 100 50,000 30 10 150 75,000 30 30 100100,000 25 25 80 75,000 35 100 190 78,000 25 100 150 60,000 15 100 160160,000 85 100 317 400,000 50 100 200 150,000 70 50 160 160,000 85 50115 150,000 70 25 60 60,000 30 100 307 315,000 100 100 216 140,000 80 50130 150,000 40 100 362 190,000 60 50 190 150,000 40 100 200 185,000 100100 387 300,000 170 50 100 130,000 20 150 588 735,000 300

We selected cells that do not express the common leukocyte antigen,CD45, or the erythroid precursor marker, glycophorin-A (GlyA).CD45⁻GlyA⁻ cells constitute 1/10³ marrow mononuclear cells. CD45⁻GlyA⁻cells were plated in wells coated with fibronectin in with 2% FCS, andEGF, PDGF-BB, dexamethasone, insulin, linoleic acid, and ascorbic acid.After 7-21 days, small clusters of adherent cells developed. Usinglimiting dilution assays, we determined that the frequency of cellsgiving rise to these adherent clusters is 1/5×10³ CD45⁻ GlyA cells.

When colonies appeared (about 10³ cells) cells were recovered bytrypsinization and re-plated every 3-5 days at a 1:4 dilution under thesame culture conditions. Cell densities were maintained between 2-8×10³cells/cm². Cell doubling time was 48-60 h. Immunophenotypic analysis byFACS of cells obtained after 10-12 cell doubling showed that cells didnot express CD31, CD34, CD36, CD38, CD45, CD50, CD62E and CD62-P, Muc18,cKit, Tie/Tek, and CD44. Cells expressed no HLA-DR or HLA-class-I andexpressed low levels of β2-microglobulin. Cells stained highly positivewith antibodies against CD10, CD13, CD49b, CD49e, CDw90, Flk1. The MASCphenotype remained unchanged for >30 cell doublings (n=15). MASCcultures with cells capable of proliferating beyond 30 cell doublingsand differentiating to all mesodermal cell-types (see below) have beenestablished from >85% of donors, age 2-50 years. In 10 donors, we haveexpanded MASC for >50 cell doublings. When cells were cultured inserum-free medium, also supplemented with 10 ng/mL IGF, cell doublingwas slower (>60 h), but >40 cell doublings could be obtained. As wasseen for cells cultured with 2% FCS without IGF, cells cultured inserum-free medium were HLA-class-I and CD44 negative, and coulddifferentiate into all mesodermal phenotypes, as described below.

When cells were plated on collagen-type-I or laminin in stead offibronectin, they expressed CD44 and HLA-DR, and could not be expandedbeyond 30 cell doublings. When EGF or PDGF were omitted cells did notproliferate and died, while increased concentrations of these cytokinesallowed initial growth of MASC but caused loss of proliferation beyond20-30 cell doublings. Addition of higher concentrations of dexamethasonealso caused loss of proliferation beyond 30 cell doubling. When cellswere cultured with >2% FCS in the culture medium they expressed CD44,HLA-DR and HLA-class-I. Likewise, culture at high density (>8×10³cells/cm²) was associated with the acquisition of CD44, HLA-DR andHLA-class-I and Muc-18, which is similar to the phenotype described forMASC. Culture at high density or with higher concentrations of FCS wasalso associated with loss of expansion capacity, and cells did notproliferate beyond 25-30 cell doublings.

We attempted to clone MASC by replating MASC at 1 cell/well oncecultures had been established. From 3 donors, we plated >2000 cellssingly in FN coated 96 well plates with the same culture medium. In nowell did we detect cell growth. Of note, when cells were deposited at 10cells/well, we found cell growth in approximately 4% of wells. Progenyof 5% of these wells could be expanded to >10⁷ cells.

Telomere length of MASC from 5 donors (age 2-50 years) cultured for 15cell doublings was between 11-16 kB. In 3 donors, this was 3 kB longerthan telomere length of blood lymphocytes obtained from the same donors.Telomere length of cells from 1 donor evaluated after 15 cell doublings,30 cells doublings and 45 cell doublings remained unchanged. Cytogeneticanalysis of MASC recovered after 30 cell doublings showed a normalkaryotype.

Example 2 Differentiation of MASCs

To induce osteoblast differentiation, serum-free medium was supplementedwith 10⁻⁷ M of dexamethasone, 10 mM ascorbic acid, and 10mM-glycerophosphate. Osteoblast differentiation was confirmed bydetection of calcium mineralization, alkaline phosphatase expression,and production of bone sialoprotein, osteopontin, osteocalcin andosteonectin, which are relatively specific for bone development (seeFIG. 7).

To induce differentiation into cartilage, serum-free medium, aspreviously described, was supplemented with 100 ng/ml TGF-1 (R&DSystems, Minneapolis, Minn.). Cells were induced to differentiate whileadherent to fibronectin, or in suspension culture, with both methodsproducing differentiated cartilage cells. Differentiation to formcartilage cells was confirmed by detection of collagen type II, as wellas the glycosaminoglycan aggrecan (see FIG. 7).

To induce adipocyte differentiation, 10⁻⁷ M dexamethasone and 100 μg/mlinsulin were added to the culture medium. Adipocyte differentiation wasalso induced by replacing serum-free medium with medium containing 20%horse serum. Adipocyte differentiation was detected by detection of LPLand aP2.

To induce skeletal myocyte differentiation, >80% confluent MASCs weretreated with either 3 μM 5-azacytidine for 24 h and then maintained inMASC medium with EGF and PDGF-BB, expression of muscle specific proteinswas seen as early as 5 days after changing culture conditions. Two daysafter induction, we detected the Myf5, Myo-D and Myf6 transcriptionfactors. After 14-18 days, Myo-D was expressed at significantly lowerlevels, whereas Myf5 and Myf6 persisted. We detected desmin andsarcomeric actin as early as 4 days after induction, and fast-twitch andslow-twitch myosin at 14 days (FIG. 7). By immunohistochemistry, 70-80%of cells expressed mature muscle proteins after 14 days. When we added20% horse serum we demonstrated fusion of myoblasts into myotubes thatwere multinucleated (FIG. 7). Of note, treatment with 5-azacytidine alsoinduced expression of Gata4 and Gata6 during the first week of culture,and cardiac troponin-T after 14 days. In addition, smooth muscle actinwas detected at 2 days after induction and persisted till 14 days.

Smooth muscle cell differentiation was when we added 100 ng/mL PDGF asthe sole cytokine to confluent MASC maintained in serum-free MASC mediumfor 14 days. Cells expressed markers of smooth muscle (Fig. x). We foundpresence of myogenin from day 4 on and desmin after 6 days. Smoothmuscle actin was detected from day 2 on and smooth muscle myosin after14 days. After 14 days, approximately 70% of cells stained positive withanti-smooth muscle actin and myosin antibodies. We could also detectMyf5 and Myf6 proteins, but not Myo-D after 2-4 days, which persistedtill day 15. (FIG. 57).

Cardiac muscle differentiation was induced by adding 100 ng/ml basicfibroblast growth factor (bFGF) to the standard serum-free culture mediapreviously described herein. Cells were confluent at onset of bFGFtreatment. To induce further development of cardiac tissues, 100 ng/ml5-azacytidine, 100 ng/ml bFGF, and 25 ng/ml bone morphogenetic proteins2 and 4 (BMP-2 and BMP-4) were added to the culture medium. Cellswere >80% confluent at onset of treatment to induce cardiac tissuedifferentiation. Gata4 and Gata6 were expressed as early as day 2 andpersisted till day 15. Expression of Myf6 and desmin was seen after day2 and myogenin after day 6. Cardiac troponin-T was expressed after day 4and cardiac troponin-I and ANP after day 11. These mature cardiacproteins were detected in >70% of cells by immunohistochemistry on day15. When the cultures were maintained for >3 weeks, cells formedsyncithia and we saw infrequent spontaneous contractions occurring inthe cultures, which were propagated over several mm distance. (FIG. 7)Again, we also detected Myf5 and myf6 and smooth muscle actin after day6.

Vascular endothelial growth factor (VEGF), at a concentration of 20ng/ml, was added to serum-free medium minus other growth factors toinduce endothelial cell differentiation by day 15-20 ex vivo.Endothelial cell differentiation was confirmed by immunofluorescencestaining to detect cellular proteins and receptors associated withendothelial cell differentiation. Results are shown in FIG. 7.

Hematopoietic differentiation was induced by culturing MASCs in collagentype IV coated wells with in PDGF-BB- and EGF-containing MASC mediumwith 5% FCS and 100 ng/mL SCF that was conditioned by the AFT024 feeder,a fetal liver derived mesenchymal line that supports murine and humanrepopulating stem cells ex vivo. Cells recovered from these culturesexpressed cKit, cMyb, Gata2 and G-CSF-R but not CD34 (RT-PCR). Becausehemopoiesis is induced by factors that are released by embryonalvisceral endoderm, we co-cultured human MASCs with βGal⁺ murine EBs inthe presence of human SCF, Flt3-L, Tpo and Epo. In 2 separate studies,we detected a small population of βGal⁻ cells that expressed human CD45

We induced “stromal” differentiation by incubating MASC with IL-1α, FCS,and horse serum. To demonstrate that these cells can supporthemopoiesis, feeders were irradiated at 2Gy and CD34⁺ cord blood cellsplated in contact with the feeder. After 2 weeks, progeny was replatedin methylcellulose assay to determine the number of colony forming cells(CFC). A 3-5-fold expansion of CFC was seen.

Confluent MASC cultures were treated with hepatocyte growth factor (HFG)and KGF. After 14 days, cells expressed MET (the HGF receptor),associated with hepatic epithelial cell development, cytokeratin 18 and19.

Example 4 Transduction of MASCs from Adult Marrow

Once MASC cultures have been established after about 3-10 subcultures,MASCs were retrovirally transduced with an enhanced green fluorescenceprotein (eGFP) containing vector on two consecutive days. Retroviralvectors that were used were the MFG-eGFP or MND-eGFP-SN constructs,kindly provided by Donald Kohn, M.D., LA Childrens Hospital, LosAngeles, Calif. Both vectors were packaged in the amphotropic cell linePA317 or the Gibon-ape leukemia packaging cell line PG13. Retroviralsupernatant was produced by incubating the producer feeder with MASCsexpansion medium for 48 hours. Supernatant was filtered and frozen at−80° C. until use. Semiconfluent MASCs were subcultured in MASCsexpansion culture medium. After 24 hours media was replaced withretrovirus containing supernatants and 8 g/mL protamine (Sigma) for 5hours. This was repeated 24 hours later. Two to three days after thelast transduction, eGFP⁺ cells were selected on a FACS Star Plus Flowcytometer with a Consort computer (all from Becton Dickinson Inc) at 10cells/well of 96 well plates coated with 5 ng/mL FN, and 40-85% ofadherent cells expressed the eGFP gene. Using the automatic celldeposition unit (ACDU) on the fluorescence activated cell sorter, 10eGFP⁺ cells per well of 96 well plates coated with fibronectin weresorted. Cells were maintained in MASCs expansion medium for 1-7 months.After 3-4 weeks, adherent cells had reached confluence in 3-4% of thewells. The cells were again culture expanded. Progeny of <1 well perplate could be expanded to generate >10⁷ cells (an additional 48 celldoublings). Thus, 1/10⁷-1/10⁸ bone marrow cells has extensiveproliferative potential.

The clonal expanded cell populations were then divided in 5-10populations. Some cells were cryopreserved undifferentiated, whereasother cells were induced to differentiate into osteoblasts,chondrocytes, stromal cells, skeletal and smooth muscle myoblasts andendothelial cells. To demonstrate differentiation along a given pathway,and to confirm tissue identity, cells were either examined byimmunohistochemistry and/or Western blot for proteins known to bepresent in the differentiated cell types.

Single cell sorting or ring cloning has been used to show single cellorigin of a cell population. However, because MASC are adherent cells itis possible that two rather than a single cell are selected by FACS orby ring cloning. The fact that integration of retroviruses is random wasused to prove clonal origin of all differentiated cells. Because of therandom viral integration, the host cell DNA that flanks the retroviralLTR is cell specific. Upon cell division, all daughter cells can beidentified based on presence of the retrovirus in the identical locationin the host cell genome.

Inverse polymerase chain reaction (PCR) was used to amplify the hostcell DNA flanking the 3 and the 5 LTR of the retroviral insert. InversePCR was done using a protocol kindly provided to us by Jan Nolta, Ph.D.,LA Children Hospital, Los Angeles, Calif. Briefly, DNA was extractedfrom undifferentiated MASC as well as from differentiated progeny, cutwith Taq1 (Invitrogen) the fragments ligated and inverse PCR performedto obtain the sequence of the 5′ flanking host cell DNA. This inversePCR technique or Southern blot analysis have extensively been used inhematopoietic stem cell biology to demonstrate that every differentiatedlineage is derived from a single cell. Once the flanking DNA had beenamplified, 200-300 bases were sequenced and primers were designed thatspecifically recognize the flanking DNA. Undifferentiated anddifferentiated cells were then subjected to PCR using one primerspecific for the flanking DNA and one primer that recognizes the 5′ longterminal repeat (LTR) to amplify DNA from the differentiated progeny.For each of the 3 samples that were examined a single cell specific DNAsequence flanking the 5′ LTR, which was identical for undifferentiatedand differentiated cells was identified. This proves single cell originof all cells of “mesodermal” origin.

Using this technique, the present studies confirm that osteoprogenitorcells exist in marrow and these cells can differentiate intoosteoblasts, chondrocytes, adipocytes, fibroblasts, and marrow stromalcells. The present inventors also demonstrate that a single marrowderived cell can give rise to cells from both splanchnic and visceralmesoderm. Further, the karyotype of cells that have been cultured formore than nine months is normal indicating that their massive expansioncapacity is due to their stem cell nature or not because of tumorgenesis or immortalization.

Example 5 Generation of Glial and Neuronal Cells from Adult Bone MarrowMesenchymal Stem Cells

Differentiated neurons are post-mitotic and little or no neuronalregeneration is observed in vivo. Therapies for neurodegenerative andtraumatic disorders of the brain may be significantly furthered if new,proliferating neural stem cells (NSC) could be introduced in thedefective areas of the brain which would resume the function of thedefective tissue. It has now been discovered that MASCs selected frompost-natal bone marrow that differentiate to all mesodermal cell typescan also differentiate to neurons, oligodendrocytes, and astrocytes.

MASC cultures were established as described in example 1. Neuraldevelopment was induced as follows. Generation of neurons, astrocytesand oligodendrocytes was done in medium consisting of neuraldifferentiation medium. This medium comprised the following: 10-95%DMEM-LG (preferably about 60%), 5-90% MCDB-201 (preferably about 40%),1×ITS, 1×LA-BSA, 10⁻⁷ to 10⁻⁹ M Dexamethasone (preferably about 10⁻⁸ M),10⁻³ to 10⁻⁵ M ascorbic acid 2-phosphate (preferably about 10⁻⁴ M) and0.5-100 ng/mL EGF (preferably about 10 ng/mL). The medium may alsocontain one or more of the following cytokines in order to inducedifferentiation into certain cell types:

-   -   5-50 ng/mL bFGF (preferably about 100 ng/mL)—astrocyte,        oligodendrocyte, neuron (type unknown));    -   5-50 ng/mL FGF-9 (preferably about 10 ng/mL)—astrocyte,        oligodendrocyte, GABAergic and dopaminergic neurons    -   5-50 ng/mL FGF-8 (preferably about 10 ng/mL)—dopaminergic,        serotoninergic and GABAergic neurons, no glial cells    -   5-50 ng/mL FGF-10 (preferably about 10 ng/mL)—astrocytes,        oligodendrocytes, not neurons    -   5-50 ng/mL FGF-4 (preferably about 10 ng/mL)—astrocytes,        oligodendrocytes but not neurons    -   5-50 ng/mL BDNF (preferably about 10 ng/mL)—Dopaminergic neurons        only)    -   5-50 ng/mL GDNF (preferably about 10 ng/mL)—GABAergic and        dopaminergic neurons    -   5-50 ng/mL CNTF (preferably about 10 ng/mL)—GABAergic neurons        only

The choice of growth factors to induce differentiation of MASCs intoneural cells was based on what is known in embryonic development of thenervous system or from studies that evaluated in vitro NSCdifferentiation. All culture medium was serum-free and supplemented withEGF, which is a strong ectodermal inducer. FGFs play a key role inneuronal development. When human post-natal marrow derived MASCs werecultured with both 100 ng/mL bFGF and 10 ng/mL EGF, differentiation toastrocytes, oligodendrocytes and neurons was seen. Astrocytes wereidentified as glial-fibrilar-acidic-protein (GFAP) positive cells,oligodendrocytes were identified as glucocerebroside positive (GalC) andneurons were identified as cells that express in a sequential fashionNeuroD, Tubulin-IIIB (Tuji), synaptophysin and neurofilament 68, 160 and200. Cells did not express markers of GAGAergic, dopaminergic orserotoninergic neurons.

FGF-9, first isolated from a glioblastoma cell line, inducesproliferation of glial cells in culture. FGF-9 is found in vivo inneurons of the cerebral cortex, hippocampus, substantia nigra, motornuclei of the brainstem and Purkinje cell layer. When cultured for 3weeks with 10 ng/mL FGF-9 and EGF MASCs generated astrocytes,oligodendrocytes and GABAergic and dopaminergic. During CNS development,FGF-8, expressed at the mid/hindbrain boundary and by the rostralforebrain, in combination with Sonic hedgehog, induces differentiationof dopaminergic neurons in midbrain and forebrain. It was found thatwhen MASCs were cultured with 10 ng/mL FGF-8 and EGF for 3 weeks bothdopaminergic and GABAergic neurons were produced. FGF-10 is found in thebrain in very low amounts and its expression is restricted to thehippocampus, thalamus, midbrain and brainstem where it is preferentiallyexpressed in neurons but not in glial cells. Culture of MASCs in 10ng/mL FGF-10 and EGF for three weeks generated astrocytes andoligodendrocytes, but not neurons. FGF-4 is expressed by the notochordand is required for the regionalisation of the midbrain. When treatedwith 10 ng/mL FGF-4 and EGF for 3 weeks MASCs differentiated intoastrocytes and oligodendrocytes but not neurons.

Other growth factors that are specifically expressed in the brain andthat affect neural development in-vivo and in-vitro include brainderived neurotrophic factor (BDNF), glial derived neurotrophic factor(GDNF) and ciliary neurotrophic factor (CNTF). BDNF is a member of thenerve growth factor family that promotes in vitro differentiation ofNSC, human subependymal cells, and neuronal precursors to neurons andpromotes neurite outgrowth of hippocamal stem cells in vivo. Consistentwith the known function of BDNF to support survival of dopaminergicneurons of the substantia nigra, when MASCs were treated with 10 ng/mLBDNF and EGF exclusive differentiation into tyrosine hydroxylasepositive neurons was seen. GDNF is a member of the TGF− superfamily. Inearly neurogenesis, GDNF is expressed in the anterior neuroectodermsuggesting that it may play a key role in neuronal development. GDNFpromotes survival of motor neurons in peripheral nerve and muscle andhas neurotrophic and differentiation abilities. It was found that GDNFinduced MASCs to differentiate into GABAergic and dopaminergic neurons.CNTF, first isolated from ciliary ganglion, is a member of the gp130family of cytokines. CNTF promotes neuronal survival early indevelopment. In embryonic rat hippocampal cultures CNTF increased thenumbers of GABAergic and cholinergic neurons. In addition, it preventedcell death of GABAergic neurons and promoted GABA uptake. CNTF exertedthe same GABAergic induction on MASCs as they differentiated exclusivelyinto GABAergic neurons after three weeks of exposure to CNTF.

Some hematopoietic cytokines have been shown to be trophic factors ofNSC, such as IL-11 and LIF, as mentioned above. In addition, in vitrostudies on neuronal precursor cells have shown that SCF, Flt3L, EPO,TPO, G-CSF, and CSF-1 act early in the differentiation of neural cellswhereas IL5, IL7, IL9, and IL11 act later in neuronal maturation. MASCsinduced with a combination of early acting cytokines (10 ng/mLThrombopoietin (kind gift from Amgen Inc., Thousand Oaks, Calif.), 10ng/mL granulocyte colony stimulating factor (Amgen), 3 U erythropoietin(Amgen) and 10 ng/mL interleukin-3 (R&D Systems), followed by culturefor 1 month in a medium conditioned by the murine fetal liver feederlayer, AFT024 (a kind gift from Dr. Ihor Lemishka, Princeton University,NJ) supplemented with 14 ng/mL fetal liver tyrosine kinase-3 ligand (akind gift from Immunex Inc, Seattle, Wash.) and 15 ng/mL SCF (a kindgift from Amgen) differentiated into astrocytes, oligodendrocytes andneurons. Neurons generated under these conditions were immature as theyexpressed neurofilament 68 but not 200.

In some cultures, MASCs had been retrovirally transduced with an eGFPcontaining vector (described in Example 4 above). Differentiated glialand neuronal cells continued to express eGFP. This indicates that thesecells can be genetically modified without interfering with theirdifferentiation. Thus, undifferentiated MASCs can generate a neural stemcell that then gives rise to astrocytes, oligodendrocytes and neurons.

The ease with which MASCs can be isolated from post-natal marrow, exvivo expanded and induced to differentiate in vitro to glial cells orspecific neuronal cell types circumvents one of the key problems in NSCtransplantation, namely the availability of suitable donor tissue.

The cells of the present invention can be used in cell replacementtherapy and/or gene therapy to treat or alleviate symptoms of congenitalneurodegenerative disorders or storage disorders such as, for instance,mucopolysaccharidosis, leukodystrophies (globoid-cell leukodystrophy,Canavan disease), fucosidosis, GM2 gangliosidosis, Niemann-Pick,Sanfilippo syndrome, Wolman disease, and Tay Sacks. They can also beused to treat or alleviate symptoms of acquired neurodegenerativedisorders such as Huntingtons, Parkinsons, Multiple Sclerosis, andAlzheimers. They can also be used for traumatic disorders such asstroke, CNS bleeding, and CNS trauma; for peripheral nervous systemdisorders such as spinal cord injury or syringomyelia; for retinaldisorders such as retinal detachment, macular degeneration and otherdegenerative retinal disorders, and diabetic retinopathy.

Example 6 Hematopoietic Development

Hematopoietic Stem Cells (HSC) are mesodermal in origin. It was longthought that HSC originate from yolk sac mesoderm. There is ampleevidence that primitive erythroid cells originate in the yolk sac. It isless clear whether definitive hemopoiesis also originates from cells inthe yolk sac. A series of recent studies in chick embryos, murine andhuman embryos have suggested that definitive hemopoiesis may be derivedfrom mesodermal cells present in the embryo proper, namely in the AGMregion. In humans, between day 22 and 35, a small population of Flk1⁺cells develops in the dorsal aorta that differentiates into CD34⁺endothelial or hemopoietic cells. It is believed that these are thecells that colonize the fetal liver. Although cells with hemopoieticpotential originate in the dorsal aorta, their differentiation andcommitment to mature hemopoietic cells requires that they migrate to theliver where the endodermal environment is conducive for hemopoieticdevelopment. In contrast, cells that remain in the AGM region will notdevelop into hemopoietic cells.

Some of the clones in the present MASC cultures have hemopoieticpotential. MASC differentiate into endothelial cells and form whatresembles embryoid bodies. These same cell aggregates differentiate intohemopoietic cells. The small, suspended aggregates were trypsinized, andreplated on FN, collagen type IV or ECM. Medium consisted either of0.5-1000 ng/mL PDGF-BB (preferably about 10 ng/mL) and 0.5-1000 ng/mLEGF (preferably about 10 ng/mL) containing MASC medium supplemented with5-1000 ng/mL SCF (preferably about 20 ng/mL) or a combination of IL3,G-CSF, Flt3-L and SCF (2-1000 ng/mL, preferably about 10-20 ng/mL).Alternatively 0.5-1000 ng/mL PDGF-BB (preferably about 10 ng/mL) and0.5-1000 ng/mL EGF (preferably about 10 ng/mL) containing MASC mediumwas used with 5% FCS and 1-1000 ng/mL SCF (preferably about 100 ng/mL)that was conditioned by AFT024 cells. Cells recovered from either ofthese cultures expressed cKit, cMyb, Gata2 and G-CSF-R(RT-PCR/immunohistochemistry) indicating that hemopoieticdifferentiation is achievable.

Example 7 Epithelial Development

Applicants have also been able to demonstrate epithelial development.Briefly, a vessel was coated with 1-100 ng/mL fibronectin along withother ECM products such as 1-100 ng/mL laminin, collagens or IV andmatrigel. The medium used comprised the following: 10-95% DMEM-LG, 5-90%MCDB-201, 1×ITS, 1×LA-BSA, 10⁻⁷-10⁻⁹ M Dexamethasone (preferably 10⁻⁸),10⁻³ to 10⁻⁵ M ascorbic acid 2-phosphate (preferably 10⁻⁴). The mediummay also contain one or more of the following cytokines

-   -   0.5-100 ng/mL EGF (preferably about 10 ng/mL)    -   0.5-1000 ng/mL PDGF-BB (preferably about 10 ng/mL)    -   0.5-1000 ng/mL HGF (hepatocyte growth factor) (preferably about        10 ng/mL)    -   0.5-1000 ng/nL KGF (keratinocyte growth factor) (preferably        about 10 ng/mL)

Some of the cells were pancytokeratin positive, and cytokeratin 18 and19 positive, which would suggest that these cells are endodermal inorigin (i.e., hepatic epithelium, biliary epithelium, pancreatic acinarycells, or gut epithelium). Some of the cells demonstrated the presenceof H-Met, or the hepatocyte growth factor receptor, which are specificfor hepatic epithelium and renal epithelium. Other cells demonstratedthe presence of keratin, which is compatible with skin epithelium.

The cells of the present invention can be used in cell replacementtherapy and/or gene therapy to treat or alleviate symptoms of severalorgan diseases. The cells could be used to treat or alleviate congenitalliver disorders, for example, storage disorders such asmucopolysaccharidosis, leukodystrophies, GM2 gangliosidosis; increasedbilirubin disorders, for instance Crigler-Najjar syndrome; ammoniadisorders such as inborn errors of the urea-cycle, for instanceOrnithine decarboxylase deficiency, citrullinemia, and argininosuccinicaciduria; inborn errors of amino acids and organic acids such asphenylketoinuria, hereditary tyrosinemia, and Alpha1-antitrypsindeficiency; and coagulation disorders such as factor VIII and IXdeficiency. The cells can also be used to treat acquired liver disordersdue to viral infections. The cells of the present invention can also beused in ex vivo applications such as to generate an artificial liver(akin to kidney dialysis), to produce coagulation factors and to produceproteins or enzymes generated by liver epithelium.

The cells of the present invention can also be used to in cellreplacement therapy and/or gene therapy to treat or alleviate symptomsof biliary disorders such as biliary cirthosis and biliary atresia.

The cells of the present invention can also be used to in cellreplacement therapy and/or gene therapy to treat or alleviate symptomsof pancreas disorders such as pancreatic atresia, pancreas inflammation,and Alpha1-antitrypsin deficiency. Further, as pancreas epithelium canbe made from the cells of the present invention, and as neural cells canbe made, beta-cells can be generated. These cells can be used for thetherapy of diabetes (subcutaneous implantation or intra-pancreas orintra-liver implantation.

Further, the cells of the present invention can also be used to in cellreplacement therapy and/or gene therapy to treat or alleviate symptomsof gut epithelium disorders such as gut atresia, inflammatory boweldisorders, bowel infarcts, and bowel resection.

Moreover, the cells of the present invention can also be used to in cellreplacement therapy and/or gene therapy to treat or alleviate symptomsof skin disorders such as alopecia, skin defects such as burn wounds,and albinism.

Example 8 Expressed Gene Profile of MASCS, Cartilage and Bone

Using Clontech and Invitrogen cDNA arrays the inventors evaluated theexpressed gene profile of human MASCs cultured at seeding densities of2×10³/cm² for 22 and 26 cell doublings. In addition the inventorsevaluated changes in gene expression when MASCs were induced todifferentiate to cartilage and bone for 2 days.

-   -   MASCs do not express CD31, CD36, CD62E, CD62P, CD44-H, cKit,        Tie, receptors for IL1, IL3, IL6, IL11, G-CSF, GM-CSF, Epo,        Flt3-L, or CNTF, and low levels of HLA-class-I, CD44-E and        Muc-18 mRNA.    -   MASCs express mRNA for the cytokines BMP1, BMP5, VEGF, HGF, KGF,        MCP1; the cytokine receptors Flk1, EGF-R, PDGF-R1α, gp130,        LIF-R, activin-R1 and -R2, TGFR-2, BMP-R1A; the adhesion        receptors CD49c, CD49d, CD29; and CD10.    -   MASCs express mRNA for hTRT, oct-4, sox-2, sox-11, sox-9, hoxa4,        -5, -9, Dlx4, MSX1, PDX1    -   Both cartilage and bone lost/had decreased expression oct-4,        sox-2, Hoxa4, 5, 9; Dlx4, PDX1, hTRT, TRF1, cyclins, cdk's,        syndecan-4; dystroglycan integrin α2, α3, β1, FLK1, LIF-R,        RAR-α, RARγ, EGF-R, PDGF-R1a and -B, TGF-R1 and -2, BMP-R1A,        BMP1 and 4, HGF, KGF, MCP1    -   Osteoblast differentiation was associated with acquisition        of/increase in expression of Hox7, hox11, sox22, cdki's,        syndecan-4, decorin, lumican, fibronectin, bone sialoprotein,        TIMP-1, CD44, β8, β5 integrin, PTHr-P, Leptin-R, VitD3-R,        FGF-R3, FGF-R2, Estrogen-R, wnt-7a, VEGF-C, BMP2    -   Cartilage differentiation was associated with acquisition of        Sox-9, FREAC, hox-11, hox7, CART1, Notch3, cdki's, collagen-II,        fibronectin, decorin, cartilage glycoprotein, cartilage        oligomeric matrix protein, MMPs and TIMPs, N-cadherin, CD44, α1        and α6 integrin, VitD3-R, BMP2, BMP7

Example 9 Characterization of Differentially Expressed Genes in MASCsvs. Osteoblasts by Subtractive Hybridization

The present inventors used a subtraction approach to identify geneticdifferences between undifferentiated MASCs and committed progeny. Poly-AmRNA was extracted from undifferentiated MASCs and cells induced todifferentiate to the osteoblast lineage for 2 days. Subtraction andamplification of the differentially expressed cDNAs was done using thePCR-Select kit from Clonetech, as per manufacturer's recommendationwithout modification. Gene sequences expressed in day 2 osteoblastcultures were analyzed, but not those in undifferentiated MASCs.

Eighty-six differentially expressed cDNA-sequences were sequenced. Itwas confirmed by Northern that the mRNAs were indeed specificallyexpressed in day 2 osteoblast progenitors and not MASCs. The sequenceswere compared (using the BLAST algorithm) to the following databases:SwissProt, GenBank protein and nucleotide collections, ESTs, murine andhuman EST contigs.

Sequences were categorized by homology: 8 are transcription factors, 20are involved in cell metabolism; 5 in chromatin repair; 4 in theapoptosis pathway; 8 in mitochondrial function; 14 are adhesionreceptors/ECM components; 19 are published EST sequences with unknownfunction and 8 are novel.

For 2 of the novel sequences, Q-RT-PCR was performed on MASCs induced todifferentiate to bone for 12 h, 24 h, 2 d, 4 d, 7 d and 14 d from 3individual donors. Genes are expressed during the initial 2 and 4 daysof differentiation respectively, and down regulated afterwards.

Genes present in undifferentiated MASCs, but not day 2 osteoblasts, werealso analyzed. Thirty differentially expressed genes have been sequencedand 5 of them are EST sequences or unknown sequences. Presence of thesegenes in MASCs but not day 2 osteoblasts is confirmed by Northern blot.

Example 10 MASC Engraftment

Studies were initiated to examine if MASCs engraft and persist in vivo.

eGFP⁺ MASCS were injected intramuscularly into NOD-SCID mice. Animalswere sacrificed 4 weeks later and muscle examined to determine if, ashas been described for human ES cells, teratomas develop. In 5/5animals, no teratomas were seen. eGFP positive cells were detected.Also, eGFP⁺ MASCS IV were infused intrauterine in fetal SCID mice.Animals were evaluated immediately after birth. PCR analysisdemonstrated presence of eGFP⁺ cells in heart, lung, liver, spleen andmarrow.

When MASCs are transplanted stereotaxically in the intact brain orinfarcted brain of rats, they acquire a phenotype compatible with neuralcells, and persist for at least 6 weeks. These studies show that humanMASCs can engraft in vivo and differentiate in an organ specific fashionwithout developing into teratomas.

The studies also show that MASCs are distinctly different than embryonicstem cells or germ cells. MASCs represent a new class of multipotentstem cells that can be derived from multiple organs of adults andchildren.

Example 11 Demonstration of the Ability to Select, Expand andCharacterize MASCs from Murine Origin

MASCs can be generated from mouse marrow and can be present in organsother than marrow.

1. Identification of MASCs in Mouse Marrow

The investigators selected MASCs from mouse marrow. Marrow from C57/BL6mice was obtained and mononuclear cells or cells depleted of CD45 andGlyA positive cells (n=6) plated under the same culture conditions usedfor human MASCs (10 ng/mL human PDGF-BB and EGF). When marrowmononuclear cells were plated, we depleted CD45⁺ cells 14 days afterinitiation of culture to remove hemopoietic cells. As for human MASCs,cultures were re-seeded at 2,000 cells/cm² every 2 cell doublings.

In contrast to what we saw with human cells, when fresh murine marrowmononuclear cells depleted on day 0 of CD45⁺ cells were plated in MASCsculture, no growth was seen. When murine marrow mononuclear cells wereplated, and cultured cells 14 days later depleted of CD45⁺ cells, cellswith the morphology and phenotype similar to that of human MASCsappeared. This suggests that factors secreted by hemopoietic cells maybe needed to support initial growth of murine MASCs. When cultured withPDGF-BB and EFG alone, cell doubling was slow (>6 days) and culturescould not be maintained beyond 10 cell doublings. Addition of 10 ng/mLLIF improved cell growth and >70 cell doublings have been obtained. Whencultured on laminin, collagen type IV or matrigel, cell growth was seen,but cells were CD44+ and HLA-class-I positive. As for human cells,C57/BL6 MASCs cultured with LIF on fibronectin coated dishes are CD44and HLA-class-I negative, stain positive with SSEA-4, and expresstranscripts for oct-4, LIF-R and sox-2.

MASCS derived from mouse marrow can be induced to differentiate intocardiac muscle cells, endothelium and neuroectodermal cells usingmethods also used to induce differentiation of human MASCs. Therefore,C57Bl6 mouse marrow derived MASCs are equivalent to those obtained fromhuman marrow.

2. MASCs are Present in Tissues Other than Marrow

The inventors examined if MASCs are present in other organs such asliver and brain. Marrow, brain or liver mononuclear cells from 5-day oldFVB/N mice, dissociated with collagenase and trypsin were plated in MASCcultures with EGF, PDGF-BB and LIF on fibronectin. 14 days later, CD45⁺cells were removed and cells maintained in MASCs culture conditions asdescribed above. Cells with morphology similar to that of human MASC andmurine MASC derived from marrow of C57/Bl6 mice grew in culturesinitiated with marrow, brain or liver cells. Cells expressed oct-4 mRNA.

The inventors also examined mice transgenic for an oct-4 promoter-eGFPgene. In these animals, eGFP expression is seen in primordial germ cellsas well as in germ cells after birth. As MASCs express oct-4, we testedwhether eGFP positive cells could be found in marrow, brain, and liverof these animals after birth. We sorted eGFP⁺ cells (1% brightestpopulation) from marrow, brain and liver from 5 day-old mice. Whenevaluated by fluorescence microscopy, <1% of sorted cells from brain andmarrow were eGFP⁺. oct-4 mRNA could be detected by Q-RT-PCR in thesorted population. Sorted cells have been plated under conditions thatsupport murine MASCs (fibronectin coated wells with EGF, PDGF, LIF).Cells survived but did not expand. When transferred to murine embryonicfibroblasts, cell growth was seen. When subsequently transferred to MASCcultures, cells with morphology and phenotype similar to that of MASCderived using classical MASC selection and culture methods from humanmarrow or marrow of C57/Bl6 or FVB/N mice were obtained.

The invention is described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin its scope. All referenced publications, patents and patentdocuments are intended to be incorporated by reference, as thoughindividually incorporated by reference.

REFERENCES

-   1. Thomson J, Kalishman J, Golos T, Durning M, Harris C, Becker R,    Hearn J: Isolation of a primate embryonic stem cell line. Proc Natl    Acad Sci USA 92:7844-8, 1995-   2. Thomson J A, Itskovitz-Eldor J, Shapiro S S, Waknitz M A,    Swiergiel J J, Marshall V S, Jones J M: Embryonic stem cell lines    derived from human blastocysts. Science 282:114-114, 1998-   3. Shamblott M, Axelman J, Wang S, Bugg E, Littlefield J, Donovan P,    Blumenthal P, Huggins G, Gearhart J: Derivation of pluripotent stem    cells from cultured human primordial germ cells. Proc Natl Acad Sci    USA 95:13726-31, 1998-   4. Williams R L, Hilton D J, Pease S, Willson T A, Stewart C L,    Gearing D P, Wagner E F, Metcalf D, Nicola N A, Gough N M: Myeloid    leukaemia inhibitory factor maintains the developmental potential of    embryonic stem cells. Nature 336:684-7, 1988-   5. Orkin S: Embryonic stem cells and transgenic mice in the study of    hematopoiesis. Int J Dev Biol 42:927-34, 1998-   6. Weissman I L: Translating stem and progenitor cell biology to the    clinic: barriers and opportunities. Science 287:1442-6, 2000-   7. Gage F H: Mammalian Neural Stem Cells. Science 287:1433-1438,    2000-   8. Svendsen C N, Caldwell M A, Ostenfeld T: Human neural stem cells:    Isolation, expansion and transplantation. Brain Path 9:499-513, 1999-   9. Okabe S, Forsberg-Nilsson K, Spiro A C, Segal M, McKay R D:    Development of neuronal precursor cells and functional postmitotic    neurons from embryonic stem cells in vitro. Mech Dev 59:89-102, 1996-   10. Potten C: Stem cells in gastrointestinal epithelium: numbers,    characteristics and death. Philos Trans R Soc Lond B Biol Sci    353:821-30, 1998-   11. Watt F: Epidermal stem cells: markers patterning and the control    of stem cell fate. Philos Trans R Soc Lond B Biol Sci 353:831, 1997-   12. Alison M, Sarraf C: Hepatic stem cells. J Hepatol 29:678-83,    1998-   13. Haynesworth S E, Barber M A, Caplan I A: Cell surface antigens    on human marrow-derived mesenchymal cells are detected by monoclonal    antibodies. Bone 13:69-80, 1992-   14. Pittenger M F, Mackay A M, Beck S C, Jaiswal R K, Douglas R,    Mosca J D, Moorman M A, Simonetti D W, Craig S, Marshak D R:    Multilineage potential of adult human mesenchymal stem cells.    Science 284:143-147, 1999-   15. Gronthos S, Zannettino A C, Graves S, Ohta S, Hay S J, Simmon P    J: Differential cell surface expression of the STRO-1 and alkaline    phosphatase antigens on discrete developmental stages in primary    cultures of human bone cells. J Bone Miner Res 14:47-56, 1999-   16. Prockop D: Marrow stromal cells as stem cells for    nonhematopoietic tissues. Science 276:71-4, 1997-   17. Jackson K, Mi T, Goodell M A: Hematopoietic potential of stem    cells isolated from murine skeletal muscle. Proc Natl Acad Sci USA    96:14482-6, 1999-   18. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E,    Stornaiuolo A, Cossu G, Mavilio F: Muscle regeneration by bone    marrow-derived myogenic progenitors. Science 279:528-30, 1998-   19. Gussoni E, Soneoka Y, Strickland C, Buzney E, Khan M, Flint A,    Kunkel L, Mulligan R: Dystrophin expression in the mdx mouse    restored by stem cell transplantation. Nature 401:390-4, 1999-   20. Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M,    Kearne M, Magner M, Isner J M: Bone marrow origin of endothelial    progenitor cells responsible for postnatal vasculogenesis in    physiological and pathological neovascularization. Circ Res    85:221-8, 1999-   21. Lin Y, Weisdorf D J, Solovey A, Hebbel R P: Origins of    circulating endothelial cells and endothelial outgrowth from blood.    J Clin Invest 105:71-7, 2000-   22. Petersen B E, Bowen W C, Patrene K D, Mars W M, Sullivan A K,    Murase N, Boggs S S, Greenberger J S, Goff J P: Bone marrow as a    potential source of hepatic oval cells. Science 284:1168-1170, 1999-   23. Theise N D, Badve S, Saxena R, Henegariu O, Sell S, Crawford J    M, Krause D S: Derivation of hepatocytes from bone marrow cells in    mice after radiation-induced myeloablation. Hepatology 31:235-40,    2000-   24. Theise N D, Nimmakayalu M, Gardner R, Illei P B, Morgan G,    Teperman L, Henegariu O, Krause D S: Liver from bone marrow in    humans. Hepatology 32:11-6, 2000-   25. Frankel M S: In Search of Stem Cell Policy. Science 298:1397,    2000-   26. Greider C: Telomeres and senescence: the history, the    experiment, the future. Curr Biol 8:178-81, 1998-   27. Reubinoff B E, Pera M F, Fong C Y, Trounson A, Bongso A:    Embryonic stem cell lines from human blastocysts: somatic    differentiation in vitro. Nat Biotech 18:399-404, 2000-   28. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D,    Chambers I, Scholer H, Smith A: Formation of pluripotent stem cells    in the mammalian embryo depends on the POU transcription factor    Oct4. Cell 95:379-91, 1998-   29. Rosfjord E, Rizzino A: The octamer motif present in the Rex-1    promoter binds Oct-1 and Oct-3 expressed by EC cells and ES cells.    Biochem Biophys Res Commun 203:1795-802, 1997-   30. Ben-Shushan E, Thompson J R, Gudas L J, Bergman Y: Rex-1, a gene    encoding a transcription factor expressed in the early embryo, is    regulated via Oct-3/4 and Oct-6 binding to an octamer site and a    novel protein, Rox-1, binding to an adjacent site. Mol Cell Biol    18:1866-78, 1998-   31. Uwanogho D, Rex M, Cartwright E J, Pearl G, Healy C, Scotting P    J, Sharpe P T: Embryonic expression of the chicken Sox2, Sox3 and    Sox11 genes suggests an interactive role in neuronal development.    Mech Dev 49:23-36, 1995-   32. Baum C, Weissman I, Tsukamoto A, Buckle A, Peault B: Isolation    of a candidate human hematopoietic stem cell population. Proc Natl    Acad Sci USA 89:2804, 1992-   33. Jordan C, McKearn J, Lemischka I: Cellular and developmental    properties of fetal hematopoietic stem cells. Cell 61:953-963, 1990-   34. Bhatia M, Wang J, Knapp U, Bonnet D, Dick J: Purification of    primitive human hematopoietic cells capable of repopulating    immune-deficient mice. Proc Natl Acad Sci USA 94:5320, 1997-   35. Goodell M, Rosenzweig M, Kim H, Marks D, DeMaria M, Paradis G,    Grupp S, Sieff C, Mulligan R, Johnson R: Dye efflux studies suggest    that hematopoietic stem cells expressing low or undetectable levels    of 34 antigen exist in multiple species. Nature Medicine    3:1337-1345, 1997-   36. Zijlmans J M, Visser J W, Kleiverda K, Kluin P M, Willemze R,    Fibbe W E: Modification of rhodamine staining allows identification    of hematopoietic stem cells with preferential short-term or    long-term bone marrow-repopulating ability. Proc Natl Acad Sci USA    92:8901-8905, 1995-   37. Phillips R L, Ernst R E, Brunk B, Ivanova N, Mahan M A, Deanehan    J K, Moore K A, Overton G C, Lemischka I R: The genetic program of    hematopoietic stem cells. Science 288:1635-40, 2000-   38. Martin G R: Isolation of a pluripotent cell line from early    mouse embryos cultured in medium conditioned by teratocarcinoma stem    cells. Proc Natl Acad Sci USA 78:7634-8, 1981-   39. Wobus A M, Holzhausen H, Jakel P, Schoneich J: Characterization    of a pluripotent stem cell line derived from a mouse embryo. Exp    Cell Res 52:212-9, 1984-   40. Kannagi R, Cochran N A, Ishigami F, Hakomori S, Andrews P W,    Knowles B B, Solter D: Stage-specific embryonic antigens (SSEA-3 and    -4) are epitopes of a unique globo-series ganglioside isolated from    human teratocarcinoma cells. EMBO J 2:2355-61, 1983-   41. Scholer H R, Hatzopoulos A K, Balling R, Suzuki N, Gruss P: A    family of octamer-specific proteins present during mouse    embryogenesis: evidence for germline-specific expression of an Oct    factor. EMBO J 8:2543-50, 1989-   42. Yuan H, Corbi N, Basilico C, Dailey L: Developmental-specific    activity of the FGF-4 enhancer requires the synergistic action of    Sox2 and Oct-3. Genes Dev 9:2635-45, 1995-   43. Rosner M H, Vigano M A, Ozato K, Timmons P M, Poirier F, Rigby P    W, Staudt L M: A POU-domain transcription factor in early stem cells    and germ cells of the mammalian embryo. Nature 345:686-92, 1990-   44. Pikarsky E, Sharir H, Ben-Shushan E, Bergman Y: Retinoic acid    represses Oct-3/4 gene expression through several retinoic    acid-responsive elements located in the promoter-enhancer region.    Mol Cell Biol 14:1026-38, 1994-   45. Niwa H, Miyazaki J, Smith A G: Quantitative expression of    Oct-3/4 defines differentiation, dedifferentiation or self-renewal    of ES cells. Nat Genet 24:372-6, 2000-   46. Cooke J E, Godin I, Ffrench-Constant C, Heasman J, Wylie C C:    Culture and manipulation of primordial germ cells. Methods Enzymol    255:37-58, 1993-   47. Hodes R J: Telomere length, aging, and somatic cell turnover. J    Exper Med 190:153-156, 1999-   48. Choi K, Kennedy M, Kazarov A, Papadimitriou J C, Keller G: A    common precursor for hematopoietic and endothelial cells.    Development 125:725-732, 1998-   49. Medvinsky A, Dzierzak E: Definitive hematopoiesis is    autonomously initiated by the AGM region. Cell 86:897, 1996-   50. Yoder M, Hiatt K, Mukherjee P: In vivo repopulating    hematopoietic stem cells are present in the murine yolk sac at day    9.0 postcoitus. Proc. Natl. Acad. Sci. USA 94:6776, 1997-   51. Spangrude G, Heimfeld S, Weissman I: Purification and    characterization of mouse hematopoietic stem cells. Science 241:58,    1988-   52. Tricot G, Gazitt Y, Leemhuis T, Jagannath S, Desikan K R, Siegel    D, Fassas A, Tindle S, Nelson J, Juttner C, Tsukamoto A, Hallagan J,    Atkinson K, Reading C, Hoffman R, Barlogie B: Collection, tumor    contamination, and engraftment kinetics of highly purified    hematopoietic progenitor cells to support high dose therapy in    multiple myeloma. Blood 91:4489-95, 1998-   53. Gothot A, Pyatt R, McMahel J, Rice S, Srour E F: Functional    heterogeneity of human CD34(+) cells isolated in subcompartments of    the G0/G1 phase of the cell cycle. Blood 90:4384-4393, 1997-   54. Goodell M, Brose K, Paradis G, Conner A, Mulligan R: Isolation    and functional properties of murine hematopoietic stem cells that    are replicating in vivo. J Exp Med 183:1797-1806, 1996-   55. McCune J M, Namikawa R, Kaneshima H, Shultz L D, Lieberman M,    Weissman I L: The SCID-hu mouse: murine model for the analysis of    human hematolymphoid differentiation and function. Science    24:1632-1639, 1988-   56. Moore K A, Hideo E, Lemischka I R: In vitro maintenance of    highly purified transplantable hematopoietic stem cells. Blood    89:4337-437, 1997-   57. Fraser C, Szilvassy S, Eaves C, Humphries R: Proliferation of    totipotent hematopoietic stem cells culture at limiting dilution on    supportive marrow stroma. Proc Natl Acad Sci USA 89:1968-1972, 1992-   58. McKay R: Stem cells in the central nervous system. Science    276:66-71, 1997-   59. Huard J M, Youngentob S L, Goldstein B J, Luskin M B, Schwob J    E: Adult olfactory epithelium contains multipotent progenitors that    give rise to neurons and non-neuronal cells. J Comp Neurol    400:469-486, 1998-   60. Palmer T D, Takahashi J, Gage F H: The adult rat hippocampus    contains primordial neural stem cells. Mol Cell Neurosci 8:389-404,    1997-   61. Lois C, Alvarez-Buylla A: Proliferating subventricular zone    cells in the adult mammalian forebrain can differentiate into    neurons and glia. Proc Natl Acad Sci USA 90:2074-7, 1993-   62. Roy N S, Wang S, Jiang L, Kang J, Benraiss A, Harrison-Restelli    C, Fraser R A, Couldwell W T, Kawaguchi A, Okano H, Nedergaard M,    Goldman S A: In vitro neurogenesis by progenitor cells isolated from    the adult human hippocampus. Nat Med 5:271-7, 2000-   63. Johansson C B, Momma S, Clarke D L, Risling M, Lendahl U, Frisen    J: Identification of a neural stem cell in the adult mammalian    central nervous system. 1998 96:25-34, 1999-   64. Fridenshtein A: Stromal bone marrow cells and the hematopoietic    microenvironment. Arkh Patol 44:3-11, 1982-   65. Wakitani S, Saito T, Caplan A: Myogenic cells derived from rat    bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle    Nerve 1417-26:18, 1995-   66. Gronthos S, Graves S, Ohta S, Simmons P: The STRO-1+ fraction of    adult human bone marrow contains the osteogenic precursors. Blood    84:4164-73, 1994-   67. Colter D C, Class R, DiGirolamo C M, Prockop D J: Rapid    expansion of recycling stem cells in cultures of plastic-adherent    cells from human bone marrow. roc Natl Acad Sci USA 97:3213-8, 2000-   68. Yui J, Chiu C, Lansdorp P: Telomerase activity in candidate stem    cells from fetal liver and adult bone marrow. Blood    91:91(9):3255-62, 1998-   69. Bjornson C, Rietze R, Reynolds B, Magli M, Vescovi A: Turning    brain into blood: a hematopoietic fate adopted by adult neural stem    cells in vivo. science 283:354-7, 1999-   70. Almeida Porada G, Crapnell H, Porada C, Benoit H, Quesenberry P,    Zanjani E D: In vivo hematopoietic potential of human neuronal stem    cells. Exp Hematol 28, Supplement 1:61 (abstract), 2000-   71. Clarke D L, Johansson C B, Wilbertz J, Veress B, Nilsson E,    Karlstrom H, Lendahl U, Frisen J: Generalized potential of adult    neural stem cells. Science 288:1660-3, 2000-   72. Rideout W M, 3rd, Wakayama T, Wutz A, Eggan K, Jackson-Grusby L,    Dausman J, Yanagimachi R, Jaenisch R: Generation of mice from    wild-type and targeted ES cells by nuclear cloning. Nat Genet    24:109-10, 2000-   73. Wilmut I, Schnieke A E, McWhir J, Kind A J, Campbell K H: Viable    offspring derived from fetal and adult mammalian cells. Nature    385:810-3, 1997-   74. Tsonis P A: Regeneration in vertebrates. Dev Biol 221:273-84,    2000-   75. Lemischka I: The power of stem cells reconsidered? Proc Natl    Acad Sci USA 96:1493-5, 1999-   76. Anderson R, Fassler R, Georges-Labouesse E, Hynes R O, Bader B    L, Kreidberg J A, Schaible K, Heasman J, Wylie C: Mouse primordial    germ cells lacking beta1 integrins enter the germline but fail to    migrate normally to the gonads. Development 126:1655-64, 1999-   77. Keller G, Snodgrass H R: Human embryonic stem cells: the future    is now. Nat Med 5:151-152, 1999-   78. Lefebvre V, de Crombrugghe B: Toward understanding SOX9 function    in chondrocyte differentiation. Matrix Biol 16:529-40, 1998-   79. Yoshida K, Chambers I, Nichols J, Smith A, Saito M, Yasukawa K,    Shoyab M, Taga T, Kishimoto T: Maintenance of the pluripotential    phenotype of embryonic stem cells through direct activation of gp130    signalling pathways. Mech Dev 45:163-71, 1994-   80. Ma Y G, Rosfjord E, Huebert C, Wilder P, Tiesman J, Kelly D,    Rizzino A: Transcriptional regulation of the murine k-FGF gene in    embryonic cell lines. Dev Biol 154:45-54, 1992-   81. Anderson R, Copeland T K, Scholer H, Heasman J, Wylie C: The    onset of germ cell migration in the mouse embryo. Mech Dev 91:61-8,    2000-   82. Gerstenfeld L C, Shapiro F D: Expression of bone-specific genes    by hypertrophic chondrocytes: implication of the complex functions    of the hypertrophic chondrocyte during endochondral bone    development. J Cell Biohem 62:1-9, 1996-   83. Binette F, McQuaid D P, Haudenschild D R, Yaeger P C, McPherson    J M, Tubo R: Expression of a stable articular cartilage phenotype    without evidence of hypertrophy by adult human articular    chondrocytes in vitro. J Orthop Res 16:207-16, 1998-   84. Cai R L: Human CART1, a paired-class homeodomain protein,    activates transcription through palindromic binding sites. Biochem    Biophys Res Commun 250:305-11, 1998-   85. Dietz U H, Sandell L J: Cloning of a retinoic acid-sensitive    mRNA expressed in cartilage and during chondrogenesis. J Biol Chem    271:3311-6, 1996-   86. Konieczny S F, Emerson C P Jr: Differentiation, not    determination, regulates muscle gene activation: transfection of    troponin I genes into multipotential and muscle lineages of 10T1/2    cells. Mol Cell Biol 5:2423-32, 1985-   87. Dinsmore J, Ratliff J, Deacon T, Pakzaban P, Jacoby D, Galpern    W, Isacson O: Embryonic stem cells differentiated in vitro as a    novel source of cells for transplantation. Cell Transplant    5:131-143, 1996-   88. Chen J, Goldhamer D: Transcriptional mechanisms regulating MyoD    expression in the mouse. Cell Tissue Res 296:213-9, 1999-   89. Wasserman S: F H proteins as cytoskeletal organizers. Cell    Biology 8:111-115, 1998-   90. Mesnard L, Samson F, Espinasse I, Durand J, Neveux J Y,    Mercadier J J: Molecular cloning and developmental expression of    human cardiac troponin T. FEBS Lett 328:139-44, 1993-   91. Doumit M E, Merkel R A: Conditions for isolation and culture of    porcine myogenic satellite cells. Tissue Cell 24:253-62, 1992-   92. Hirschi K K, Rohovsky S A, D'Amore P A: PDGF, TGF-beta, and    heterotypic cell-cell interactions mediate endothelial cell-induced    recruitment of 10T1/2 cells and their differentiation to a smooth    muscle fate. J Cell Biol 141:805-14, 1998-   93. Miano J, Cserjesi P, Ligon K, Periasamy M, Olson E: Smooth    muscle myosin heavy chain exclusively marks the smooth muscle    lineage during mouse embryogenesis. Circ Res 75:803-12, 1994-   94. Wobus A M, Kaomei G, Shan J, Wellner M C, Rohwedel J, Ji G,    Fleischmann B, Katus H A, Hescheler J, Franz W M: Retinoic acid    accelerates embryonic stem cell-derived cardiac differentiation and    enhances development of ventricular cardiomyocytes. J Mol Cell    Cardiol 29:1525-39, 1998-   95. Laverriere A C, MacNeill C, Mueller C, Poelmann R E, Burch J B,    Evans T: GATA-4/5/6, a subfamily of three transcription factors    transcribed in developing heart and gut. J Biol Chem 269:23177-84,    1994-   96. Bhavsar P K, Dhoot G K, Cumming D V, Butler-Browne G S, Yacoub M    H, Barton P J: Developmental expression of troponin I isoforms in    fetal human heart. FEBS Lett 292:5-8, 1991-   97. Forssmann W, Richter R, Meyer M: The endocrine heart and    natriuretic peptides: histochemistry, cell biology, and functional    aspects of the renal urodilatin system. Histochem Cell Biol    110:335-57, 1998-   98. Punzel M, Wissink S, Miller J, Moore K, Lemischka I, Verfaillie    C: The myeloid-lymphoid initiating cell (ML-IC) assay assesses the    fate of multipotent human progenitors in vitro. blood 93:3750-6,    1999-   99. Thiemann F T, Moore K A, Smogorzewska E M, Lemischka I R, Crooks    G M: The murine stromal cell line AFT024 acts specifically on human    CD34+CD38− progenitors to maintain primitive function and    immunophenotype in vitro. Exp Hematol 26:612-619, 1998-   100. Rosenberg J B, Foster P A, Kaufman R J, Vokac E A, Moussalli M,    Kroner P A, Montgomery R R: Intracellular trafficking of factor VIII    to von Willebrand factor storage granules. J Clin Invest 101:613-24,    1998-   101. Baumhueter S, Dybdal N, Kyle C, Lasky L: Global vascular    expression of murine CD34 a sialomucin-like endothelial ligand for    L-selectin. Blood 84:2554, 1994-   102. Hamagushi I, Huang X L, Takakura N, Tada J, Yamagushi Y, Kodama    H, Suda T: In vitro hematopoietic and endothelial cell development    from cells expressing TEK receptor in murine aorta-gonad-mesonephros    region. Blood 93:1549-1556, 1999-   103. Shalaby F, Ho J, Stanford W, Fischer K, Schuh A, Schwartz L,    Bernstein A, Rossant J: A requirement for Flk1 in primitive and    definitive hematopoiesis and vasculogenesis. Cell 89:981-90, 1997-   104. Newman P: The biology of PECAM-1. J Clin Invest 99:3, 1997-   105. Tedder T, Steeber D, Chen A, Engel P: The selectins: vascular    adhesion molecules. FASEB J 9:866, 1995-   106. Nishikawa S, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H:    Progressive lineage analysis by cell sorting and culture identifies    FLK1+VE-cadherin+ cells at a diverging point of endothelial and    hemopoietic lineages. Development 125:1747-57, 1998-   107. Belaoussoff M, Farrington S M, Baron M H: Hematopoietic    induction and respecification of A-P identity by visceral endoderm    signaling in the mouse embryo. Development 125:5009-18, 1988-   108. Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, Peterson A C,    Reynolds B A: Multipotent CNS stem cells are present in the adult    mammalian spinal cord and ventricular neuroaxis. J Neurosci    16:7599-609, 1996-   109. Shihabuddin L S, Ray J, Gage F H: FGF-2 is sufficient to    isolate progenitors found in the adult mammalian spinal cord. Exp    Neurol 148:577-86, 1997-   110. Ciccolini F, Svendsen C N: Fibroblast growth factor 2 (FGF-2)    promotes acquisition of epidermal growth factor (EGF) responsiveness    in mouse striatal precursor cells: Identification of neural    precursors responding to both EGF and FGF-2. J Neuroscience    18(19):7869-7880, 1998-   111. Julien J, Mushynski W: Neurofilaments in health and disease.    Prog Nucleic Acid Res Mol Biol 61:1-23, 1998-   112. Schaafsma H, Ramaekers F: Cytokeratin subtyping in normal and    neoplastic epithelium: basic principles and diagnostic applications.    Pathol Annu 29:21-62, 1994-   113. Lazaro C A, Rhim J A, Yamada Y, Fausto N: Generation of    hepatocytes from oval cell precursors in culture. Cancer Res    58:5514-22, 1998-   114. Kiem H, Heyward P, Winkler A, Potter J, Allen J, Miller A,    Andrew R: Gene transfer into marrow repopulating cells: comparison    between amphotropic and gibbon ape leukemia virus pseudotyped    retroviral vectors in a competitive repopulation assay in baboons.    Blood 90:4638-45, 1997-   115. Nolta J, Dao M, Wells S, Smogorzewska E, Kohn D: Transduction    of pluripotent human hematopoietic stem cells demonstrated by clonal    analysis after engraftment in immune-deficient mice. Proc Natl Acad    Sci USA 93:2414-9, 1996-   116. Huibregtse B A, Johnstone B, Goldberg V M, Caplan A I: Effect    of age and sampling site on the chondro-osteogenic potential of    rabbit marrow-derived mesenchymal progenitor cells. Orthop Res    18:18-24, 2000-   117. Bandyopadhyay P, Ma X, Linehan-Stieers C, Kren B, Steer C:    Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA    oligonucleotides. Targeted delivery of liposomes and    polyethyleneimine to the asialoglycoprotein receptor. J Biol Chem:    10163-72, 1999-   118. Sielaff T D, Nyberg S L, Rollins M D, Hu M Y, Amiot B, Lee A,    Wu F J, Hu W S, Cerra F B: Characterization of the three-compartment    gel-entrapment porcine hepatocyte bioartificial liver. Cell Biol    Toxicol 13:357-64, 1997-   119. Peshwa M V, Wu F J, Sharp H L, Cerra F B, Hu W S: Mechanistics    of formation and ultrastructural evaluation of hepatocyte spheroids.    32:197-203, 1996-   120. Rogler L E: Selective bipotential differentiation of mouse    embryonic hepatoblasts in vitro. Am J Pathol 150:591-602, 1997-   121. Block G D, Locker J, Bowen W C, Petersen B E, Katyal S, Strom S    C, Riley T, Howard T A, Michalopoulos G K: Population expansion,    clonal growth, and specific differentiation patterns in primary    cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a    chemically defined (HGM) medium. J Cell Biol 132:1133-49, 1996-   122. Hao Q L, Thiemann F T, Petersen D, Smogorzewska E M, Crooks G    M: Extended long-term culture reveals a highly quiescent and    primitive human hematopoietic progenitor population. Blood    88:3306-3313, 1996-   123. Visser J W, Bol S J, van den Engh G: Characterization and    enrichment of murine hemopoietic stem cells by fluorescence    activated cell sorting. Exp Hematol 9:644-55, 1981-   124. Gothot A, van der Loo J C, Clapp D W, Srour E F: Cell    cycle-related changes in repopulating capacity of human mobilized    peripheral blood CD34(+) cells in non-obese diabetic/severe combined    immune-deficient mice. Blood 92:2641-9, 1998-   125. Klug M G, Soonpaa M H, Koh G Y, Field L J: Genetically selected    cardiomyocytes from differentiating embryonic stem cells form stable    intracardiac grafts. J Clin Invest 98:216-24, 1996-   126. Kipriyanov S M, Little M: Generation of recombinant antibodies.    Mol Biotechnol 12:173-201, 1999-   127. Shinohara N, Demura T, Fukuda H: Isolation of a vascular cell    wall-specific monoclonal antibody recognizing a cell polarity by    using a phage display subtraction method. Proc Natl Acad Sci USA    97:2585-90, 2000-   128. Iyer V R, Eisen M B, Ross D T, Schuler G, Moore T, Lee J C F,    Trent J M, Staudt L M, Hudson J J, Boguski M S, Lashkari D, Shalon    D, Botstein D, Brown P O: The transcriptional program in the    response of human fibroblasts to serum. Science 283:83-7, 1999-   129. Scherf U, Ross D T, Waltham M, Smith L H, Lee J K, Tanabe L,    Kohn K W, Reinhold W C, Myers T G, Andrews D T, Scudiero D A, Eisen    M B, Sausville E A, Pommier Y, Botstein D, Brown P O, Weinstein J N:    A gene expression database for the molecular pharmacology of cancer.    Nat Biotech 24:236-44, 2000-   130. Alizadeh A A, Eisen M B, Davis R E, Ma C, Lossos I S, Rosenwald    A, Boldrick J C, Sabet H, Tran T, Yu X, Powell J I, Yang L, Marti G    E, Moore T, Hudson J J, Lu L, Lewis D B, Tibshirani R, Sherlock G,    Chan W C, Greiner T C, Weisenburger D D, Armitage J O, Warnke R,    Staudt L M: Distinct types of diffuse large B-cell lymphoma    identified by gene expression profiling. Nature 403:503-11, 2000-   131. Diehn M, Eisen M B, Botstein D, Brown P O: Large-scale    identification of secreted and membrane-associated gene products    using DNA microarrays. Nat Biotech 25:58-62, 2000-   132. Wang E, Miller L D, Ohnmacht G A, Liu E T, Marincola F M:    High-fidelity mRNA amplification for gene profiling. Nat Biotechnol    18:457-9, 2000-   133. Somia N V, Schmitt M J, Vetter D E, Van Antwerp D, Heinemann S    F, Verma I M: LFG: an anti-apoptotic gene that provides protection    from Fas-mediated cell death. Proc Natl Acad Sci USA 96:12667-72,    1999-   134. Elefanty A G, Begley C G, Metcalf D, Barnett L, Kontgen F, Robb    L: Characterization of hematopoietic progenitor cells that express    the transcription factor SCL, using a lacZ “knock-in” strategy. Proc    Natl Acad Sci USA 95:11897-902, 1998-   135. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H,    Inai Y, Silver M, Isner J: VEGF contributes to postnatal    neovascularization by mobilizing bone marrow-derived endothelial    progenitor cells. EMBO J 18:3964-72, 1999-   136. Robbins P, Skelton D, Yu X, Halene S, Leonard E, Kohn D:    Consistent, persistent expression from modified retroviral vectors    in murine hematopoietic stem cells. Proc Natl Acad Sci (USA)    95:10182-87, 1998-   137. Case S, Price M, Jordan C, Yu X, Wang L, Bauer G, Haas D, Xu D,    Stripecke R, Naldini L, Kohn D, Crooks G: Stable transduction of    quiescent CD34(+)CD38(−) human hematopoietic cells by HIV-1-based    lentiviral vectors. Proc Natl Acad Sci USA 96:2988-93, 1999-   138. Uchida N, Sutton R, Friera A, He D, Reitsma M, Chang W, Veres    G, Scollay R, IL. W: HIV, but not murine leukemia virus, vectors    mediate high efficiency gene transfer into freshly isolated G0/G1    human hematopoietic stem cells. Proc Natl Acad Sci USA 95:11939-44,    1998-   139. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M,    Magner M, Isner J M, Asahara T: Ischemia- and cytokine-induced    mobilization of bone marrow-derived endothelial progenitor cells for    neovascularization. Nat Med 5:434-8, 1999-   140. Phinney D G, Kopen G, Isaacson R L, Prockop D J: Plastic    adherent stromal cells from the bone marrow of commonly used strains    of inbred mice: variations in yield, growth, and differentiation. J    Cell Biochem 72:570-85, 1999-   141. Svendsen C N, Skepper J, Rosser A E, ter Borg M G, Tyres P,    Ryken T: Restricted growth potential of rat neural precursors as    compared to mouse. Brain Res Dev Brain Res 99:253-8, 1997-   142. Cheshier S H, Morrison S J, Liao X, Weissman I L: In vivo    proliferation and cell cycle kinetics of long-term self-renewing    hematopoietic stem. Proc Natl Acad Sci USA 96:3120-5, 1999-   143. Horner P J, Power A E, Kempermann G, Kuhn H G, Palmer T D,    Winkler J, Thal L J, Gage F H: Proliferation and differentiation of    progenitor cells throughout the intact adult rat spinal cord. J    Neurosci 20:2218-28, 2000-   144. Randall T D, Weissman I L: Phenotypic and functional changes    induced at the clonal level in hematopoietic stem cells after    5-fluorouracil treatment. Blood 89:3596-606, 1997

1. An isolated multipotent mammalian stem cell that is surface antigennegative for CD44, CD45, and HLA Class I and II. 2-63. (canceled)